Total Synthesis of
Demethyllavendamycin Amides
An Honors 499 Thesis
By
Jennifer S Lucas
-
Mentor
Dr. Mohammad Behforouz
Ball State University
Muncie, IN
May 5, 2000
Expected Bate of Graduation: May 6,2000
1
Ie Abstract
This thesis involves the total synthesis of three analogs of lavendamycm, a
chemotherapeutic agent: 7-N-chloroacetyldemethyllavendamycin Amide, 7-Nchloroacetyldemethyllavendamycin Amide ofPyrrolidine, and 7-N-
-
butyryldemethyllavendamycin Amide of Piperazine. This research is part of an
ongoing project involving synthesis and structure-activity relationship studies of
various analogs of lavendamycin. These analogs contain the amide functional
group in the C-2' position. The C-2' amides have proved to be very active anticancer agents.
-,
"-
2
,-
Table Of Contents
I. Abstract
1
II. Acknowledgements
3
ill. Background Information
4
IV. Biological Activity
7
V. Total Synthesis
9
VI.
Experimental
A. General Information
B. Solvent Purification
C. Procedures
8-Hydroxy-2-methyI-5, 7-dinitroquinoline
5, 7 -Dibutyramido-8~butyroxy-2-methylquinoline
7-Butyramido-2-methylquinoline-5,8-dione
7-Butyramido-2-formylquinoline-5,8-dione
5,7 -Bis(Chloroacetamido)-8-hydroxy-2-methylquinoline
7-Chloroacetamido-2-methylquinoline-5,8-dione
7-Chloroacetamido-2-formylquinoline-5,8-dione
Tryptophan Amide
Benzyloxycarbonyltryptophan Succinimide Ester
Benzyloxycarbonyltryptophan Amide ofPyrrolidine
Tryptophan Amide ofPyrrolidine
Benzyloxycarbonyltryptophan Amide of Piperazine
Tryptophan Amide of Piperazine
7-N-Chloroacetyldemethyllavendamycin Amide
7-N-Chloroacetyldemethyllavendamycin Amide ofPyrrolidine
7-N-Butyryldemethyllavendamycin Amide of Piperazine
-
14
14
15
15
15
16
17
18
18
19
20
20
21
22
23
23
24
25
26
Appendix A
NMR / Mass Spectroscopy
27
AppendixB
Research Presentations
28
VII. Works Cited
29
3
II. Acknowledgments
I would like to express my deep gratitude and great appreciation to Dr.
Mohammad Behforouz for allowing me to participate in his research project. Dr.
Behforouz has been a teacher, mentor, and friend, as well as a great influence in my life.
Working on this research project has been an invaluable learning experience, and it could
not have been possible without the guidance and instruction of Dr. Behforouz. I am truly
thankful for the opportunity to work with such a fine individual.
I would also like to express my gratitude to Wen Cai for her daily help in the
-
laboratory. She has provided me with much knowledge and beneficial research skills.
Her patience and self-less attitude have helped my research immensely. She has been a
true friend both inside and outside of the laboratory.
I would like to thank the Ball State University Chemistry Department for the
opportunity to do research. I feel the research program is one of the best attributes of the
Chemistry Department, and I would like to commend the department for this. I would
also like to thank the National Institute of Health, the American Cancer Society, and Eli
Lilly for their continually funding of this project.
Finally, I would like to express my appreciation and gratitude to my parents for
their continual support throughout my undergraduate career.
-
4
III. Background Information
Lavendamycin (1) was discovered in the fermentation broth of the
microorganism Steptomyces lavendulae by researchers at Bristol Laboratories in 1981.1
Lavendamycin closely resembled a previously discovered antitumor agent, streptonigrin
(2), in both structure and biological activity. Studies have shown that both lavendamycin
and streptonigrin exhibit potent antitumor and anticancer activities, but are unusable thus
far due to their high cytotoxicity and low solubility. 2,3,4
o
4
1
10'
The exact mechanism for antitumor activity and cytotoxicity exhibited by
lavendamycin and streptonigrin is unknown. The quinone structure alone has shown
activities such as DNA cleavage, which is related to the quinone reduction potential. 5
Other proposed mechanisms deal with the effects of the compound on the electron
transport system within the mitochondria.6-10 No single mechanism has been able to
explain the cytotoxic activity of these compounds thus far.
.-
Lavendamycin analogs have exhibited encouraging antitumor activities against
K
ras oncogenic tumors. l1 ,12 Although lavendamycin has proven to be less potent than the
5
-
compound streptonigrin, lavendamycins are more potent than their quinolinedione
analogs (A-B ring portion oflavendamycin).7 The additional three ring system (C-E) in
lavendamycin that make up the pentacyclic structure moderates the cytotoxic effects of
the quinoline structure and increase the overall antitumor activity of the system compared
to the corresponding quinolinedione.
For necessary structure-activity relationship studies to be preformed, an efficient
method of synthesis for lavendamycin had to be developed. After the discovery of
lavendamycin in 1981, several research groups began studying the synthesis of the
compound. Kende and Ebetino at the University of Rochester published the first total
synthesis oflavendamycin in 1984. 13,14 This synthesis involved the use of a Friedlander
condensation to produce the A-B portion of the compound, and a Bischler-Napieralski
-
cyclodehydration to complete the pentacyclic structure. 13 In 1985 Hibino and group
reported a total synthesis oflavendamycin methyl ester through the use ofa PictetSpengler condensation between J3-methyl-tryptophan and a quinoline analog to produce a
pentacyclic intermediate. 15 This intermediate was modified to the finallavendamycin
product through a series of transformations. Also in 1985, Boger's research group
reported a total synthesis pathway containing twenty steps and an overall yield of less
than 1%.18
In 1993, Behforouz and group at Ball State University reported a total synthesis of
lavendamycin methyl ester with five steps and an overall yield of33%.16
-
6
-
1993 Synthesis Scheme
0
+
ACNH
Br
2
0
0
~I
e~1
heat
b-Si~
..
.
ACNH
3
CH3
0
0
Se02
...
xylene
+
ACNH
CHO
0
S
4
0
-
0
~
lN~~N
ACNH
0
C02C~
~S04H20
roDe
Q
...
""'N#
ACNH
~N
0
CH3
6
C~
7
This synthesis involved using a novel azadiene Diels-Alder reaction followed by a PictetSpengler condensation between a formylquinolinedione and a f3-methyl tryptophan.
Behforouz's highly concise procedure is much more practical than the previously
reported synthesis routes due to the reduced number of intermediates, intermediate
-
...
reflux
stabilities, and the increased overall yield. 16
7
-
In 1996, Behforouz's group reported a new method of synthesis oflavendamycin
methyl ester with an overall yield of 40%.11,17 This new method of efficient synthesis has
allowed for the production of many lavendamycin analogs such as the ones involved in
this thesis. With the ability for efficient synthesis, the analogs of the antitumor agent
lavendamycin are readily produced for structure-activity relationship tests to be
performed. These studies focus on finding a direct relationship between the biological
activities of the antitumor agents and their structure.
IV. Biological Activity
The efficient synthesis ofLavendamycin developed by Behforouz's group, has
allowed for profound structure-activity relationship studies on a variety of analogs. The
--
analogs ofLavendamycin consist of five main positions for the addition or deletion of
functional groups as shown below.
Rl=H
C~CO
HCO
~CH2CO
etc.
R2=H
Cl
OC-"3
>-
R3 =CON<
COOR
C&OR
C&OH
COOH
R5 =H
OH
F
OC~
8
Structure activity relationship studies have shown that an acetyl group at the C-7
position exhibits the most selective toxicity against tumor cells compared to parent cells.
Several other groups such as the butyryl also exhibit biological activity and selective
toxicity against rask oncogene transformed cells. The SAR studies have also shown that
an amide or ester functional group is needed at the C-2' position for increased biological
activity.
The purpose of this thesis project was to synthesize three analogs with different
substituents at the C-7 and C-2' positions to analyze the activity against oncogene
transformed cells, the solubility of the compound, and the selectivity of the compound.
All three of the analogs synthesized for this thesis involve an amide functional group in
the C-2' position. Amides in this position have shown good activity when tested against
-
different tumor lines, therefore we have synthesized three different amides in this project.
Pyrrolidine was chosen because of the high activity and selectivity it has previously
exhibited, and piperazine was chosen for increased water solubility. The extra N-H on
the piperazine is a position available for making a salt to increase solubility. In the C-7
position, two compounds synthesized exhibit a chloroacetyl group, and one compound
exhibits a butyryl group. The chloroacetyl group was added to compare the biological
activity of the analog when adding a chloro group verses an acetyl group. The butyryl
group in the C-7 position was chosen because of the increased anti-tumor activity it has
exhibited. A comparison of these compounds with other amide analogs previously
synthesized should provide additional information on the role of the substituents in
relation to activity and solubility.
9
v.
Total Synthesis of Lavendamycin Analogs
Results and Discussion
The Pictet-Spengler condensation of a quinolinedione aldehyde and a tryptophan
results in the formation of the pentacyclic structure oflavendamycin. The quinolinedione
aldehyde and tryptophan are fully functionalized prior to the condensation, as well as
purified and dried. The Pictet-Spengler condensation is believed to involve a
spiroindolenine intermediate. 19
Preparation of the quinoline aldehyde proceeds in the manner diagramed below
(refer to Scheme 1). The starting compound, 8-hydroxy-2-methylquinoline 1 is
commercially available, and a nitration yielded a dinitro product 2. The nitration is
-
accomplished using a mixture of concentrated nitric and sulfuric acids. The nitro groups
add in an ortho-para fashion because of the hydroxyl group on the starting compound.
The reaction is exothermic, and therefore performed in an ice bath.
Scheme 1
}h-PdlC
H20 ,HCl
NBOCR
~
(RCO)zO
RCOHNy~~
NaOACINa:z~
OH
-
~C~~
HOAc
WI~ __
o
•
RCOHN
W~
O
SeO:z
_ _....
_
N"'-:: CIfJ
o
38,3b
t
RCOHN
48, 4b
0
R(b)=OCH2
N"" CHO
58,5b
-
10
The next step involves the reduction of the dinitro compound 2 to an ammonium
chloride salt. This reaction is performed on the Parr Hydrogenator at a pressure of 43 psi
utilizing H2 over 5% palladium charcoal as a catalyst. The nitro groups are reduced to
amino groups, and then HCI changes these amino groups to ammonium chloride salts.
Since amino compounds are easily oxidized, the ammonium salts act as protecting
groups. The ammonium chloride salt compound was not isolated. Instead, the reaction
mixture with the salt was immediately placed into a solution with sodium acetate, sodium
sulfite, and either chloroacetic anhydride or butyric anhydride. The sodium acetate acted
as a base, and the sodium sulfite acted as a base and an antioxidant. This reaction is a
nucleophilic attack of the amino groups to the carbonyl carbons of the anhydride,
resulting in the dibutrylamido (3a) or dichloroacetamido (3b) compounds.
-
Compound 3 was then oxidized using potassium dichromate, in a solution of
glacial acetic acid and water, to form the quinolinedione 4. The reaction mixture was
extracted with dichloromethane, and the organic phase was neutralized with 3%
NaHC03. The quinolinedione 4, was then further oxidized with selenium dioxide in a
solution of wet dioxane. The water acts to catalyze the reaction, forming a reactive oxide
of selenium. This oxidizes the methyl group of the quinolinedione to an aldehyde. The
quinoline aldehyde 5 is used in the Pictet-Spengler condensation to form the final
lavendamycin product.
The preparation of tryptophan amide 7 involves a simple neutralization of the
commercially available tryptophan amide hydrochloride 6, purchased from Aldrich (refer
to scheme 2). The hydrochloride salt of the tryptophan amide was neutralized by the
-
dropwise addition of 14% ammonium hydroxide to form the tryptophan 7.
11
--
Scheme 2
14%NBtOH
o
Pictel-Spengl",
Condensation
5b
fr;
CI~OCHNAy~A-:r
N
o
-
H
~
I
8
The preparation of the tryptophan amide of pyrrolidine and the tryptophan amide
of piperazine was performed in the following manner (refer to Scheme 3). N-Cbz
tryptophan 9, purchased from Aldrich, was reacted with N-hydroxysuccinimide in the
presence of dicyclohexylcarbodiimide. This reaction involved an esterification, yielding
Cbz tryptophan succinimide ester 10. This reaction involved the formation of urea,
which was difficult to remove. The reaction mixture was allowed to sit over two nights
in the refrigerator to allow the urea to precipitate out of solution. Compound 10 was
reacted with either pyrrolidine or piperazine in the presence of triethylamine to afford the
Cbz-tryptophan amide ofpyrrolidine 11a or the Cbz-tryptophan amide of piperazine lIb.
-
The Cbz protecting group of the amide was then cleaved off in a deprotection reaction to
12
yield the fully functional tryptophan amide 12. This reaction involved the use of
ammonium formate in the presence of 10% palladium on charcoal and a argon balloon.
The reaction mixture containing compound 12a was washed 3% NaHCO)
solution after the reaction was complete and the palladium on charcoal was filtered off to
remove the ammonium formate from the solution. The water layer was extracted with 5
X 20 mL dichloromethane, and the pure Tryptophan amide ofpyrrolidine was removed to
the organic phase. Since the ammonium formate remained in the aqueous phase, the
resulting compound was much cleaner than previous procedures. The deprotection
reaction ofCbz-tryptophan amide of piperazine (llb) required a large amount of catalyst
was well as a hydrogen balloon to help the reaction go to completion. The extra amino
group on the piperazine ring acted to poison the catalyst, thus a 4: 1 ratio was utilized.
-
Compound 12b is very soluble in water, therefore washing with 3% NaHCO) resulted in
a huge loss of compound. Instead, the ammonium formate was sublimed off at high
temperatures on the rotary evaporator and vacuum pump. The NMR of Tryptophan
amide of piperazine was also very difficult to interpret. The compound is totally
insoluble in CDCh, so DMSO-c4 was used as the solvent. The peaks that correspond to
the hydrogens on the piperazine ring come at the same ppm as the water peak, therefore
the ring hydrogens were difficult to interpret.
The Pictet-Spengler condensation was utilized to produce the finallavendamycin
analogs. Ana10g 8 involved the condensation of 7-Chloroacetamido-2-formylquinoline5,8-dione (Sb) and tryptophan amide (7) in anisole. The reaction mixture was heated at
155°C over a period of20 hours, and the pure compound 8 fell out of solution (49"/0).
-
The NMR of this compound exhibited two extra singlets at 7.5 ppm and 8.0 ppm due to
13
-
the chloro in the C-7 position, as well as DMSO-~ as the solvent (the compound is
insoluble in CDCh). Analog 13a involved the condensation of7-Chloroacetamido-2formylquinoline-5,8-dione (5b) and tryptophan amide ofpyrrolidine (12a) in anisole.
The reaction mixture was heated at 155°C over a period of21 hours. This reaction is
very sensitive, as the hydrolyzed form of the compound is also formed during the
reaction. A column was utilized to separate these two forms of the compound, yielding
pure 7-N-Chloroacetyldemethyllavendamycin Amide ofPyrrolidine (20%).
Schemel
~0T>
DCC
N-hydroxysucdnimide
Amine
triethylamine
c-rrNIl
(X)):
~
~
I \
N
0
NHCbz
10
H
o
r""'y"i-
R
~, ~HCbz
H
1111, 11b
HCOONl:\
o
~-R
~, ~H2
H
1211, 12b
+
RCotW
CHO
CoacieDsation
12.,12b
5a,Sb
R(a)=-NQ
-
13.,13b
14
-
VI. Experimental
A. General Information
Reagents:
8-hydroxy-2-methylquinoline, selenium dioxide, L-tryptophan amide
hydrochloride salt, N-carbobenzloxytryptophan, N-hydroxysuccinimide,
and N-dicyclohexylcarbodiimide were purchased from the Aldrich
Chemical Company.
Solvents:
All solvents utilized in the reactions were reagent grade (except for 1,4dioxane, chloroform, ethanol, and methanol which were dried before use,
see below).
NMR Spectra:
IH NMR Spectra were recorded on a Varian Gemini 200 Spectrometer
and an JEOL Eclipse 400 Spectrometer using CDCh and DMSO.
Low and High Resolution Mass Spectra:
--
Thin-Layer Chromatography:
EI and F AB Mass Spectra were obtained at
the Chemistry Department of the
University of Illinois.
Eastman silica gel strips with fluorescent indicator
were used to determine purity of all products.
B. Solvent Purification
The solvent used in the oxidation reaction to the aldehyde and in the preparation of
Cbz-tryptophan succinimide ester, 1,4-dioxane, was dried and distilled before use. The
solvent was refluxed with sodium spheres for 2-3 hours (until the spheres appeared
metallic), and then benzophenone was added to the mixture. 1,4-dioxane has a tendency
to polymerize, thus this procedure also purified the solvent.
Chloroform, absolute ethanol, and methanol were all utilized in the preparation of the
tryptophan amide derivatives. These reactions required that the solvents be completely
dry prior to use. The solvents were refluxed for several hours with calcium hydride (until
the mixture appeared milky white) and then distilled.
15
c.
Procedures
Preparation of 8-Hydroxy-2-methyl-5,7-dinitroquinoline (2)
A solution of concentrated nitric acid (210 mL) and concentrated sulfuric acid (90 mL)
was placed in a 500 mL Erlenmeyer flask equipped with a magnetic stir bar in an ice
bath. Commercially available 8-Hydroxy-2-methyquinoline (1; 40.60 g, 0.25 mol) was
added to the flask drop wise over a period of 45 minutes while the mixture was being
stirred. A brownish-yellow solution resulted, and red fumes were emitted. The solution
was then allowed to stir for 2 hours in an ice-bath. After 2 hours, the solution was poured
into a 2 L Erlenmeyer flask containing 1 L of ice water while still stirring. A bright
yellow precipitate was immediately formed. The precipitate was then filtered and
washed with ice water (500 mL) and diethyl ether (300 mL). The bright yellow product
was pressed dry with paper towel and allowed to air dry overnight. The reaction yielded
45.03 g (72%) of8-Hydroxy-2-methyl-5,7-dinitroquinoline (2). IH NMR (CDCh): 8
9.63 (1H, d, J=8.9 Hz, C-4H), 9.20 (1H, s, C-6H), 8.13 (lH, d, J=8.9 Hz, C-3H), 2.92
(3H, s, C-2CIIJ).
Preparation of 5,7-Dibutyramido-2-methyl-8-butyroxyquinoline (3a)
To a thick-walled 500 ml hydrogenation bottle, was added finely ground 8-Hydroxy-2methyl-5,7-dinitroquinoline (2; 5.98 g, 0.24 mol) and 10% HCI solution (100 ml). Next,
2.0 g of 5% Pd/C was added as a catalyst. The solution was then hydrogenated on a Parr
Hydrogenator overnight at a pressure of 30 psi. When the reaction was completed the
mixture was filtered and washed with water. The filtrate was then placed in a 1000 ml
-
round-bottomed flask equipped with a magnetic stir bar. To this solution was added
16
sodium sulfite (18 g), sodium acetate (24 g), and butyric anhydride (97.5 ml) as quickly
as possible. The stirred solution immediately formed a white precipitate, and it was then
allowed to stir for 3 hours. The precipitate was filtered off and washed with 800 ml water
until the orange tint was gone. The white product (3a) was allowed to air dry overnight
and yielded 9.82 g (80.%). IHNMR (DMSO-~):
a 9.94 (lH, s, C-7NH), 9.65 (1H, s, C-
5NH), 8.24 (1H, s, C-6H), 8.21 (lH, d, J=8.8 Hz, C-4H), 7.37 (lH, d, J=8.8 Hz, C-3H),
2.70 (2H, t, J=8.0 Hz, OCOCHzCH2CH3), 2.58 (3H, s, C-2CHJ), 2.30-2.40 (4H, m,
2NHCOCHzCH2CHJ), 1.73-1.89 (2H, m, OCOCH2CHzCHJ), 1.52-1.72 (4H, m,
2NHCOCHzCHzCHJ), 1.08 (3H, t, J=8.0 Hz, OCOCH2CHzCH 3), 0.96 (3H, t, J=8.0 Hz,
NHCOCH2CH2CHJ), 0.92 (3H, t, J=8.0 Hz, NHCOCH2CH2CHJ).
Preparation of 7-Butyramido-2-metbylquinoline-5,8-Dione (4a)
In a 1000 mL round-bottomed flask containing a magnetic stir bar, 5,7-Dibutyramido-2methyl-8butyroxyquinoline (3a; 3.29 g, 8.25 mmol) was added along with glacial acetic
acid (122 mL). While stirring vigorously, a solution of Potassium dichromate (8.80 g,
0.03 mol) and distilled water (115 mL) was added. After 2 hours, dichloromethane (70
mL) was added. This two-phase solution was then poured into a separatory funnel, and
the organic layer was removed into a 1000 mL Erlenmeyer flask. The water layer was
extracted with 12 X 50 mL dichloromethane, and saturated NaCI solution (50 mL) was
added for the last 5 extractions. The organic layer was a reddish-orange color, and the
water layer was very dark red. The combined organic layers were then washed with 3 X
200 mL 3% sodium bicarbonate solution. The organic layer was kept, and the water
-
layer was washed with 2 X 50 mL dichloromethane. The new organic phase was
17
extracted with 200 mL 3% sodium bicarbonate, and then added to the previous organic
layer. The organic solution was dried over anhydrous magnesium sulfate and rotary
evaporated to a red-orange precipitate (4a). After drying overnight on the vacuum pump,
the reaction yielded 1.56 g (73%). IH NMR (CDCh): 08.32 (1H, s, C-7NB), 7.92 (lH,
s, C-6H), 7.55 (1H, d, J=8.1 Hz, C-3H), 2.79 (3H, s, C-2C~), 2.50 (2H, t, J=7.8 Hz, C7NHCOCH1CH2CH3), 1.64-1.82 (2H, m, C7-NHCOCH2CH1CH3), 1.0 (3H, t, J=7.8 Hz,
C-7NHCOCH2CH2CH3 ).
Preparation of 7-Butyramido-2-formylquinoline-5,8-dione (5a)
To a 25 mL I round-bottomed flask equipped with a reflux condenser, argon balloon, and
magnetic stir bar was added, 7-Butyramido-2-methylquinoline-5,8-dione (4a; 0.516 g, 2
-.
mmol), selenium dioxide (0.3104 g, 2.8 mmol), 12 mL dried, distilled dioxane, and 0.25
mL water. The reaction mixture was heated at 120-130 °c and monitored by TLC. The
reaction was completed after 25 hours. To the final reaction mixture was added 12 mL
dioxane, and then was refluxed for an additional 10 minutes. The mixture was then
filtered hot on a Buchner funnel with the aid of a pipette. The filter cake and remaining
selenium were then refluxed in 10 mL dichloromethane for 10 minutes and filtered off.
The combined filtrates were then rotary evaporated until nearly dry. The reddish-brown
precipitate was dissolved in dichloromethane and washed with 2 X 50 mL 3% sodium
bicarbonate. The organic layers were saved, and the water layers were extracted with 3 X
50 mL dichloromethane. The combined organic phases were then dried over magnesium
sulfate. The bright yellow filtrate was rotary evaporated to yield 0.21 g (390/0) pure
bright yellow product (5a). IH NMR (CDCh): 0 10.31 (lH, s, C-2CHO), 8.65 (1H, d,
18
-.
J=7.68 Hz, C-4H), 8.40 (lH, s, C-7NH), 8.32 (lH, d, J=8.1 Hz, C-3H), 8.08 (1H, s, C6H), 2.54 (2H, t, J=7.34 Hz, C-7NHCOCH1CH2CH3), 1.62-1.80 (2H, m, C7NHCOCH2CH2CH3), 1.04 (3H, t, J=7.37 Hz, C-7NHCOCH2CH2CHJ).
Preparation of 5,7-Bis(Chloroacetamido)-8-hydroxy-2-methylquinoline (3b)
To a thick-walled 500 mL hydrogenation bottle was added finely ground 8-Hydroxy-2methyl-5,7-dinitroquinoline (2; 5.25 g, 0.21 mol) and 10010 HCl solution (l00 ml). Next,
2.0 g of 5% PdlC was added as a catalyst. The solution was then hydrogenated on a Parr
Hydrogenator overnight at a pressure of30 psi. When the reaction was completed the
mixture was filtered and the filter cake was washed with water (20 mL). The filtrate was
then placed in a 250 mL round-bottomed flask equipped with a magnetic stir bar. To this
-
solution was added sodium sulfite (12 g), sodium acetate (16 g), and chloroacetic
anhydride (65 g) as quickly as possible. Heat was evolved and it took several minutes for
the chloroacetic anhydride to dissolve. The reaction mixture was then stirred for one
hour during which a precipitate was formed. The mixture was then added to 100 mL of
ice water and filtered. The filter cake was then washed with cold ethanol (10 mL). The
filtrate was allowed to stand over night, and the additional precipitate was filtered. The
reaction yielded 3.61 g (50%) pure product (3b). IHNMR (DMSO-~): 0 10.19 (lH, s,
C-5NH), 9.92 (lH, s, C-7NH), 8.16 (1H, s, C-6H), 8.15 (1H, d, J=9.16Hz, C-4H), 7.43
(1H, d, J=8.8Hz, C-3H), 4.47 (2H, s, CHCH1Cl), 4.38 (2H, s, COCH1CI) ,2.71 (3H, s,
C-2CH 3).
-
19
Preparation of 7-Cbloroacetamido-2-metbylquinoline-5,8-dione (4b)
In a 500 mL round-bottomed flask equipped with a magnetic stir bar,S, 7-
Bis(Chloroacetamido)-8-hydroxy-2-methylquinoline (3b; 3.42 g, 0.01 mol) was dissolved
in glacial acetic acid (122 mL). To this solution Potassium dichromate (8.8 g, 0.03 mol)
in distilled water (115 mL) were added. This dark brown solution was stirred vigorously
overnight at room temperature with a watch glass covering the top of the flask. The
solution was then extracted with 12 X 50 mL dichloromethane. The organic extracts
were washed with 2 X 200 mL 3% sodium bicarbonate solution and added together. The
solution was dried over anhydrous magnesium sulfate and rotary evaporated to yield a
bright yellow solid. After vacuum drying the pure product weighed 1.64 g (62%). IH
NMR (CDCh): 39.54 (1H, s, C-7NH), 8.32 (1H, d, J=16.88 Hz, C-4H), 7.90 (1H, s, C6H), 7.57 (1H, d, J=8.04 Hz, C-3H), 4.24 (2H, s, COCH1Cl), 2.77 (3H, s, C-2CH3).
Preparation of 7-Cbloroacetamido-2-formylquinoline-5,8-dione (5b)
In a two-neck 25 mL round-bottomed flask equipped with a magnetic stir bar, water-
cooled condenser, and an argon balloon was added 7-Chloroacetamido-2methylquinoline-5,8-dione (4b; 0.529 g, 2 mmol), selenium dioxide (0.332 g, 3 mmol),
dried, distilled dioxane (12 mL), and distilled water (0.32 mL). The reaction mixture was
stirred to a reflux over a two hour period. The reaction was monitored by TLC, and after
17 hours it was completed. An additional 12 mL dioxane was added to the reaction
mixture and refluxed for an additional 10 minutes. Next, the solution was vacuum
filtered hot using pipettes. The filter cake was then added back to the reaction flask and
-
refluxed for 10 minutes with 10 mL dichloromethane and filtered. The filtrates were
20
,-
placed on the rotary evaporator until dry. The dry precipitate was dissolved in 50 mL
dichloromethane and placed in a separatory funnel. The mixture was then washed with 2
X 50 mL 3% sodium bicarbonate solution. The water layers were extracted with 3 X 50
mL dichloromethane, and all the organic layers were combined and dried over anhydrous
magnesium sulfate. The solution was rotary evaporated to a yellow-brown solid
weighing 0.25 g (45%). IHNMR (CDCh): 0 10.29 (1H, s, C-2CHO), 9.56 (1H, s, C-
7NH), 8.64 (lH, d, J=1.12 Hz, C-4H), 8.33 (1H, d, J=8.08 Hz, C-3H), 8.05 (lH, s, C6H), 4.26 (2H, s, COCH2CI).
Preparation of Tryptophan Amide (7)
In a 125 mL Erlenmeyer flask was added commercially available tryptophan amide
hydrochloride salt (6; 0.4794 g, 2 mmol) and 35 mL ethyl acetate. To this cloudy
solution was added 14% ammonium hydroxide solution dropwise until the solution was
clear. The resulting solution was placed in a separatory funnel, and the water layer was
separated from the organic layer. The organic layer was then washed with 5 X 6 mL
saturated sodium chloride solution. The organic layer was dried over anhydrous
magnesium sulfate, and rotary evaporated to a thick gel. The reaction mixture was
allowed to dry several days on the vacuum pump (89010). IH NMR (CDCh): 0 8.05 (1H,
s, -NH), 7.68 (1H, d, J=8.2 Hz, C-4H), 7.39 (lH, d, J=8.5 Hz, C-7H), 7.07-7.22 (2H, m,
C-2H C-5H, C-6H), 5.32 (2H, s, CONH1), 3.71 (lH, dd, J=8.1 Hz, J=7.9 Hz, C-l'H),
2.84-3.42 (2H, m, C-3'H), 1.4 (2H, s, C-2'NH1).
21
-
Preparation of Benzyloxycarbonyltryptopban Succinimide Ester (10)
In a 25 mL round-bottomed flask equipped with a magnetic stir bar and an argon filled
balloon, N-carbobenzyloxytryptophan (9; 0.338 g, Immol), N-hydroxysuccinimide
(0.155g,1 mmol) and 5 mL dried, distilled dioxane were added. The reaction mixture
was stirred in a 12°C ice bath until the solution was clear. To this clear solution was
added N-dicyclohexylcarbodimide (0.206g, 1 mmol), and a white precipitate formed after
1 minute. The reaction mixture was stirred at 15°C to 20°C for 2 hours, and then stirred
for 2 additional hours at room temperature. The reaction flask was then placed in the
refrigerator over 2 nights. The solid was filtered and rinsed with 5 mL dioxane. The
filtrate was then rotary evaporated until a thick colorless gel was formed. The product
was placed on the va.cuum pump for two days where it foamed up into a white solid (10)
-
weighing 0.4215 g (970/0). IH NMR (CDCh): 08.09 (IH, s, -NH), 7.54 (IH, d, J=7.72
Hz, C-4H), 7.32-7.37 (6H, m, C-7H, Ct;Hs), 7.19 (lH, t, J=7.5 Hz, C-5H), 7.09 (lH, t,
J=7.32 Hz, C-6H), 5.5.11-5.21 (3H, m, CHCOOCHl), 5.05 (lH, d, J=12.48 Hz, C2'NH), 3.5 (2H, m, C-3'H), 2.85 (4H, s, COCHlCHl ).
Preparation of Benzyloxycarbonyltryptopban Amide of Pyrrolidine (Ua)
In a 100 mL round-bottomed flask equipped with a magnetic stir bar and an argon filled
balloon, benzyloxycarbonyltryptophan succinimide ester (10; 0.87 g, 2 mmol),
pyrrolidine (0.166 mL, 2 mmol), dried, distilled triethylamine (0.28 mL, 2 mmol), dried,
distilled absolute ethanol (26 mL), and dried, distilled chloroform (24 mL), were added.
The reaction mixture was allowed to stir at room temperature, while being monitored by
-
TLC. The reaction was complete after 30 minutes. The reaction mixture was then rotary
22
-
evaporated to dry. This solid was dissolved in 3 X 60 mL ethyl acetate. This solution
was allowed to sit overnight in the refrigerator so the salts from the triethylamine could
precipitate out of solution. The solution was immediately filtered when removed from
the refrigerator, and the filtrate was washed with 60 mL of distilled water. The organic
phase was then washed with 2 X 60 mL 10% citric acid solution, followed by 30 mL 1 N
NaHCOJ solution. The neutralized organic phases were then washed with 5 X 10 mL
brine solution and dried over anhydrous sodium sulfate overnight. The solution was
rotary evaporated to a thick gel and placed on the vacuum pump overnight where it
foamed up into a white solid weighing 0.6935 % (89010). IH NMR (CDCh): B 7.97 (1R,
s, -NH), 7.62 (1R, d, J=8.04 Hz, CAB), 7.32-7.35 (5R, m, C6Hs), 7.29 (1R, d, J=5.84
Hz, C-7H), 7.17 (1R, t, J=7.5 Hz, C-5H), 7.10 (1R, t, J=7.72 Hz, C-6H), 7.07 (1R, s, C2H), 5.74 (1H, d, J=8.4 Hz, C-2'NH), 5.10 (2H, s, COOCH2 ), 4.69-4.73 (1H, m, C-2'H),
3.19-3.37 (5R, m, C-3' cis to the C-l amide group, -pyrrolidine-C-2H, C-5H), 2.49-2.53
(1R, m, C-3'H trans to the C-l amide group), 1.26-1.62 (4H, m, -pyrrolidine-C-3H, C4H).
Preparation of Tryptophan Amide of Pyrrolidine (12a)
In a 50 mL two necked, round-bottomed flask equipped with a stir bar and an argon filled
balloon were added benzyloxycarbonyltryptophan amide ofpyrrolidine (11a; 0.336 g,
0.86 mmol) and dried, distilled methanol (17.5 mL). To this stirred suspension, vacuum
dried ammonium formate (0.1595 g, 2.53 mmol) and 10% palladium on charcoal (0.1595
g) were added. The reaction was allowed to stir at room temperature and was monitored
-
by TLC. The reaction was found to be complete after 30 minutes. The reaction mixture
23
was then filtered and the palladium on charcoal filter cake was washed with 10 mL
methanol. The filtrate was then rotary evaporated to concentrate (about one half the
volume). To this concentrated organic solution was added 15 mL 3% NaHC03 solution,
forming a white precipitate. The solution was then extracted with 5 X 20 mL
dichloromethane. The organic phase was dried overnight on anhydrous sodium sulfate,
and it was rotary evaporated to dry yielding 0.1836 g (83%) of pure compound 12a. IH
NMR (CDCh): 08.01 (1H, s, -NH), 7.59 (1H, d, J=7.7 Hz, C-4H), 7.35 (lH, d, J=8.04
Hz, C-7H), 7.19 (lH, t, J=7.52 Hz, C-5B), 7.11 (tH, t, J=7.68 Hz, C-6B). 7.08 (lH, s, C2B), 3.99 (2H, s, -NHl), 3.82 (1H, t, J=6.96 Hz, C-2'H), 2.86-3.48 (6H, m, C-3'H,pyrrolidine-C-2Bl, C-5Bl), 1. 67 -1. 95 (4H, m, -pyrrolidine-C-3Hl , C-4Bl).
Preparation of Benzyloxycarbonyltryptophan Amide of Piperazine (llb)
In a 100 mL round-bottomed flask equipped with a magnetic stir bar and an argon filled
balloon, benzyloxycarbonyltryptophan succinimide ester (10; 1.007 g, 2.31 mmol),
piperazine (0. 1949, 2.31 mmol), dried, distilled triethylamine (0.32 mL, 2.31 mmol),
dried, distilled absolute ethanol (26 mL), and dried, distilled chloroform (24 mL), were
added. The reaction mixture was allowed to stir at room temperature, while being
monitored by TLC. The reaction was complete after 4 hours. To the reaction mixture
was added 10010 citric acid solution drop wise until a pH of 6 was reached. This solution
was then neutralized to a pH of 7-8 with 3% NaHC03 solution (added drop wise). The
neutralized reaction mixture was then washed with 3 X 2 mL brine solution and dried
over anhydrous sodium sulfate overnight. The solution was rotary evaporated to a thick
-
gel and placed on the vacuum pump where it foamed up yielding 0.7268 g (72%) of the
24
-
pure compound lla. IH NMR (DMSO-~): 0 10.89 (1H, s, -NH), 7.62 (1H, d, J=9.88
Hz, C-4H), 7.51 (1H, d, J=13.6, C-7H), 7.29-7.35 (5H, m, -C6 H5), 7.15 (lH, s, C-2H),
7.06 (1H, t, J=7.68 Hz, C-5H), 6.96 (lH, t, 8.04 Hz, C-6H), 4.99 (3H, s, NHCOOCHl -),
4.35 (lH, s, C-2'H), 3.04-3.33 (4H, m, -piperazine-C-2H, C-6H), 2.90-3.08 (2H, m, C3'H), 2.59 (4H, s, -piperazine-C-3H, C-4H), 2.34 (1H, s, -piperazine-NH).
Preparation of Tryptopban Amide of Piperazine (12b)
In a 50 mL two necked, round-bottomed flask equipped with a stir bar and an argon filled
balloon were added benzyloxycarbonyltryptophan amide of piperazine (l1b~ 0.4065 g, 1
mmol) and dried, distilled methanol (25 mL). To this stirred suspension, vacuum dried
ammonium formate (0.189 g, 3 mmol) and 10010 palladium on charcoal (0.472 g, 4 mmol)
were added. After all of the reactants were added to the flask, the argon balloon was
replaced by a hydrogen balloon. The reaction was allowed to stir at room temperature
and was monitored by TLC. The reaction was complete after 1.5 hours. The palladium
on charcoal was then filtered off, and the filter cake was washed with 10 mL methanol.
The filtrate was then rotary evaporated to dry, and left on the rotary evaporator for an
hour at a high temperature (90°C) to sublime the ammonium formate. The product, 12b,
was then placed on the vacuum pump at a temperature of 40°C for two days yielding
0.241 g (89%). IHNMR (DMSO-~): 0 10.85 (1H, s, -NH), 7.48 (lH, d, J=7.36 Hz, C4H), 7.32 (lH, d, J=6.6, C-7H), 7.14 (lH, s, C-2H), 7.06 (lH, t, J=6.22 Hz, C-5H), 6.97
(1H, t, J=7.14 Hz, C-6H), 4.04 (3H, m, C-2'H, NHl ), 3.04-3.33 (4H, m, -piperazine-C2H, C-6H), 2.90-3.08 (2H, m, C-3'H), 2.59 (4H, s, -piperazine-C-3H, C-4H), 2.34 (1H,
-
s, -piperazine-NH).
25
-
Preparation of 7-N-Chloroacetyldemethyllavendamycin Amide (8)
In a 250 mL 3 necked round-bottomed flask equipped with a magnetic stir bar, argon
flow, and a dean stock condenser, 7-Chloroacetamido-2-formylquinoline-5,8-dione (5b~
0.082569 g, 0.3 mmol), tryptophan amide
(7~
0.0609 g, .03 mmol), and anisole (120 mL)
were added. The reaction mixture was allowed to heat to 155°C over the course of three
hours and was monitored by nc. TLC showed the reaction complete after 20 hours.
The reaction mixture was allowed to cool to room temperature, and it was then filtered to
give a solid yellow precipitate. The precipitate was washed with petroleum ether and
allowed to dry overnight on the vacuum pump. The reaction yielded 0.0677g (49%) pure
compound (8). IHNMR (DMSO-c4): 012.26 (1H, s, 8'-NH), 10.19 (1H, s, C7-NH),
-
9.93 (1H, s, C-7NH), 9.75 (2H, s, CONH2), 9.12 (1H, d, J=9.16 Hz, C-4B), 9.02 (1H, s,
C-3'B), 8.61 (1H, d, J=8.8 Hz, C-3D), 8.49 (1H, d, J=8.08 Hz, C-12'B), 8.49 (1H, s, C6B), 7.82 (1H, d, J=8.04 Hz, C-9'B), 7.70 (1H, t, J=8.4 Hz, C-lO'B), 7.39 (lH, t, J=8.4
Hz, C-l1 'D), 4.55 (2H, s, CICB2CO).
Preparation of7-N-Chloroacetyldemethyllavendamycin Amide of Pyrrolidine (13a)
In a 100 mL 3 necked round-bottomed flask equipped with a magnetic stir bar, argon
flow, and a dean stock condenser were added, 7-Chloroacetamido-2-formylquinoline-5,8dione (5b; 0.04247 g, 0.1525 mmol), tryptophan amide ofpyrrolidine (12a; 0.03925 g,
0.1525 mmol), and anisole (55 mL). The reaction was heated to 155°C over a period of 3
hours. The reaction was monitored by nc and found to be complete after 18 hours.
--
The reaction mixture was then filtered hot, and the filter cake was washed with 4 mL
26
-
dichloromethane and 4 mL chloroform. The filtrate was rotary evaporated to dry.
Column chromatography was needed to purify the filter cake and the filtrate. The
product was dissolved in 20 mL dichloromethane and 4 mL methanol, and the developing
solvent was ethyl acetate. After column chromatography, the reaction yielded 0.011 g
pure product (13a) (21%). IH NMR. (DMSO-c4): 0 11.87 (1H, s, 8'-NH), 10.65 (lH, s,
C-7NH), 8.94 (lH, d, J=8.4 Hz, C-4H), 8.88 (1H, S, C-3'H), 8.57 (1H, d, J=8.04 Hz, C3H), 8.48 (1H, d, J=8.04 Hz, C-12'H), 7.81 (lH, s, C-6H), 7.71 (2H, d, t, C-9'H, Cll'H), 7.41 (lH, t, J=8.1 Hz, C-lO'H), 4.66 (2H, s, CICH2CO), 3.98 (2H, s, -pyrrolidineC-2H), 3.64 (2H, s, -pyrrolidine-C-5H), 1.96 (4H, s, -pyrrolidine-C-3H2, C-4H2).
Preparation oC7-N-butryldemethyllavendamycin Amide oCPiperazine (13b)
In a 50 mL 3 necked round-bottomed flask equipped with a magnetic stir bar, argon flow,
and a dean stock condenser were added, 7-Butrylamido-2-formylquinoline-5,8- dione
(Sa; 0.02721 g, 0.1 mmol) and anisole (35 mL). This solution was heated to 70°C, until
all of the starting compound (Sa) had dissolved. To this reaction mixture was added
tryptophan amide of piperazine (12b; 0.02724 g, 0.1 mmol) dissolved in 0.83 mL dried,
distilled pyridine. The reaction mixture was then heated to 155°C over a period of3
hours. The reaction was monitored by TLC and was stopped after 26 hours. The reaction
mixture was then filtered hot, and the filter cake was washed with 4 mL ethyl acetate and
4 mL petroleum ether. The product was impure, and column chromatography was
needed for purification.
-.
27
-
Appendix A
Index of Spectra
8-Hydroxy-2-methyl-5,7-dinitroquinoline ............................. eH NMR) ......... (2)
5,7-Dibutyramido-8-butyroxy-2-methylquinoline..................... eH NMR) ......... (3a)
7-Butyramido-2-methylquinoline-5,8-dione ......................... eH NMR) ......... (4a)
7-Butyramido-2-formylquinoline-5,8-dione ............................ eH NMR) ......... (5a)
5,7 -Bis(Chloroacetamido)-8-hydroxy-2-methylquinoline ............ eH NMR) ......... (3b)
7-Chloroacetamido-2-methylquinoline-5,8-dione ...................... eH NMR) ......... (4b)
7-Chloroacetamido-2-formylquinoline-5,8-dione ......... , ............ eH NMR) ......... (5b)
Tryptophan Amide .............. , ...... , ....... , ............................ eH NMR) ......... (7)
Benzyloxycarbonyltryptophan Succinimide Ester ..................... eH NMR) ......... (10)
Benzyloxycarbonyltryptophan Amide ofPyrrolidine .................. eHNMR) ...... (l1a)
Tryptophan Amide of Pyrrolidine ......................................... eH NMR) ...... (12a)
Benzyloxycarbonyltryptophan Amide of Piperazine ................... eH NMR) ...... (l1b)
Tryptophan Amide of Piperazine ................................... " ... eH NMR) ......... (12b)
7-N-Chloroacetyldemethyllavendamycin Amide ...................... eH NMR) ......... (8)
(Mass Spec) .... , .. (8)
7-N-Chloroacetyldemethyllavendamycin Amide ofPyrrolidine ... eH NMR) ......... (13a)
-
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28
Appendix B - Research Presentations
The author of this thesis presented the research conducted for this paper on two
different occasions. One presentation was for the Ball State University Summer
Research Program. The other presentation was at the Indiana Academy of Science
Annual Meeting November 4, 1999 in Evansville, Indiana.
--
-
29
VII. Works Cited
1) Doyle, TW.; Balitz, D.M.; Grulich, R.E.; Nettleton, D.E.; Gould, S.1.; Tann, C.H.; Meows,
AE. "Structure Determination of Lavendamycin: A New Antibiotic From Streptomyces
lavendulae.", Tetrahedron Lett., 1981, 22,4595.
2) Balitz, D.M.; Bush, lA; Bradner, W.T; Doyle, TW.; O'Herron, F.A; Nettleton, D.E..
"Isolation of Lavendamycin: A New Antibiotic From Streptomyces lavendulae", J
Antibiotic, 1982, 35, 259.
3) Hackethal, C.A; Golbey, R.B.; Tan, C.TC.; Kamofsky, D.A; Burchenal, lH .. "Clinical
Observation on the Effects of Streptonigrin in Patients with Neoplastic Disease", Antibiot.
Chemother., 1961, 11, 178.
4) Wilson, W.L.; Labra, c.; Barrist, E., "Preliminary Observation on the Use of Streptonigrin as
an Antitumor Agent in Human Beings", Antibiot. Chemother., 1961. 11, 147.
-
5) Shaikh, I.A; Johnson, F.; GroHman, AP .. "Streptonigrin. I. Structure-Activity Relationships
Among Simple Bicyclic Analogues. Route Dependence of DNA Degradation on Quinone
Reduction Potential", J Med Chem., 1986,29, 1329.
6) Bachur, N.R.; Gordon, S.L.; Gee, M.V., "A General Mechanism for Microsomal Activation of
Quinone Anticancer Agents to Free Radicals", Cancer Res., 1978,38, 1745.
7) Boger, D.L.; Yasuda, M.; Mitscher, L.; Drake, S.D.; Kitos, P.A, "Streptonigrin and
Lavendamycin Partial Structures. Probes for the Minimum, Potent Pharmacore of
Streptonigrin, Lavendamycin and Synthetic Quinoline-5,8-diones", J Aled Chem., 1987,
30,1918
8) Lown, lW.; Sim, S.-K., "Studies Related to Antitumor Antibiotics. Part VIII. Cleavage of
DNA by Streptonigrin Analogues and Relationship to Antineoplastic Activity", Can. J
Biochem., 1976,5-1,446.
9) Cone, R.; Hasan, S.K.; Lown, lW.; Morgan, AR.; "The Mechanism of the Degradation of
DNA by Streptonigrin", Can. J Biochem., 1976,5-1,219.
10) Lown, lW.; Sim, S.-K.; Chen, H.-H., "Hydroxyl Radical Production by Free and DNABound Aminoquinone Antibiotics and its Role in DNA Degradation. Electron Spin
Resonance Detection of Hydroxyl Radicals and by Spin Trapping", Call. J Biochem.,
1978, 56, 1042.
-
30
11) Behforouz, M.; Merriman, RL., "Lavendamycin Analogs and Methods and Making and
Using Them", U.S. Patent 5,525,611, June 11, 1996.
12) Behforouz, M. Grant Proposal to the NIH, 1997, unpublished.
13) Kende, AS.; Ebetino, F.H., "The Regiospecific Total Synthesis of Lavendamycin Methyl
Ester", Tetrahedron Lett., 1984,25, 923.
14) Kende, AS.; Ebetino, F.H.; Battista, R; Lorah, D.P.; Lodge, E., "New Tactics in
Heterocyclic Synthesis", Heterocycle, 1984,21, 91.
15) Hibino, S.; Okazaki, M.; Ichikawa, M.; Sato, K; Ishizu, T. Heterocycles, 1985,23,261.
16) Behforouz, M.; Gu, Z.; Cai, W.; Hom, M.A; Ahmadian, M., "A Highly Concise Synthesis of
Lavendamycin Methyl Ester", J Org. Chem., 1993,58, 7084.
-
17) Behforouz, M.; Haddad, 1.; Cai, W.; Arnold, M.A; Mohammadi, F.; Sousa, AC.; Hom,
M.A, "Highly Efficient and Practical Syntheses of Lavendamycin Methyl Ester and
Related Quinolinediones", J Org. Chem., 1996,61, 6552.
18) Boger, D.L.; Duff, S.R; Panek, M.; Yasuda, M., "Inverse Electron Demand Diels-Alder
Reactions of Heterocyclic Azadienes. Studies on the Total Synthesis of Lavendamycin:
Investigative Studies on the Preparation of the DCE-f3-Carboline Ring System and AB
Quinoline-5,8-quinone Ring System", J, Org. Chem., 1985,50, 5782.
19) Ungemach, F.; Cook, 1.M., "The Spiroindolenine Intermediate, A Review", Heterocycles,
1978, 9, 1089.
20) Tolstikov. VV.; Holpne Kozlova, N.W.; Oreshinka, T.D.; Ospiova, T.V; Preobrazhanskaza,
M.N.; Sztaricskai, F.; Balzarini, 1.; Declercq, E., "Ami des of Antibiotic Streptonigrin and
Amino Dicarboxylic Acids or Aminosugars; Synthesis and Biochemical Evaluation", J
Antibiol., 1992, -15, 1020.
-