ABSTRACT

MILLER, MARIA ROMAINE Synthesis and Application of Nine Novel Analogs of Trypsin Modulating Oostatic Factor (TMOF) in the Mosquito, Aedes aegypti. (Advised by Russell J. Linderman) The search continues for an environmentally safe, low cost, fast acting insecticide for

mosquitoes. Mosquitoes have been the cause of large-scale epidemics, such as yellow

fever, dengue fever, and dengue hemorrhagic fever over the centuries. Even now, due to

the increase in the Aedes aegypti population, the threat still exists. In this research, six of

nine compounds were synthesized as potential analogs of the decapeptide TMOF

(trypsin modulating oostatic factor), which has already been identified as a growth

inhibitor in mosquitoes. The nine compounds were derived from three different starting

materials and six were completed in eight or fewer steps. These compounds were tested

for larvacidal activity by feeding studies using Aedes aegypti. The compound 7-(3,4-

dimethoxyphenyl)-4-heptenoic acid resulted in the lowest LC50, 0.62mM.

Figure 1. Photo of Aedes aegypti mosquito

SYNTHESIS AND APPLICATION OF NINE NOVEL ANALOGS OF TRYPSIN MODULATING OOSTATIC FACTOR (TMOF)

IN THE MOSQUITO, Aedes aegypti

by

MARIA ROMAINE MILLER

A thesis submitted to the Graduate Faculty of North Carolina State University

In partial fulfillment of the Requirements for the Degree of

Master of Science

PHYSICAL AND MATHEMATICAL SCIENCES

Raleigh

2000

APPROVED BY:

___________________________________ _________________________________

_________________________

Chair of Advisory Committee

DEDICATION

First, last, and always, I give thanks and praise to God for my endless blessings. This

work is dedicated in memory of my Father and to my Mother. Everything that I have

accomplished is all to their credit. The constant support, love, and assistance from my

husband and son have made the past two years bearable. Thank You!

BIOGRAPHY

Maria Miller was raised in Jarrettsville, Maryland, where she lived until attending the

College of Notre Dame in Baltimore. There, she earned her baccalaureate degree in

chemistry. During her three years at Notre Dame, Maria served as a cadet in the Loyola

College Reserve Officer Training Corps (ROTC). She spent two additional years at the

University of Maryland and earned a second bachelor's degree in chemical engineering.

After her first year at College Park, Maria was commissioned as a Second Lieutenant in

the United States Army and served in the Maryland Army National Guard. Upon

graduation, Maria became an active duty chemical officer.

After initial chemical officer training at Fort McClellan, Alabama, Maria served as a

battalion chemical officer and decontamination platoon leader in South Korea. She was

promoted to First Lieutenant and assigned to Fort Bragg, North Carolina. Her duty

positions included: Brigade Chemical Officer, Company Executive Officer, Detachment

Commander, Regiment Chemical Officer, Battalion Adjutant, and Company Commander.

She was promoted to Captain while serving as a Detachment Commander. Maria's

military schools include: Chemical Officer Basic and Advanced Courses, Airborne

School, Advanced Airborne School, Joint Psychological Operations Staff Planners

Course, Psychological Operations Officer Course, and the Regional Studies Course. She

has been awarded: the Meritorious Service Medal, Army Commendation Medal, Army

Achievement Medal, National Defense Service Ribbon, Overseas Service Ribbon,

Airborne Badge, and the Argentine Airborne Badge. After completing all required duties

ii

for Captain, Maria was selected for the Advanced Civil Schooling Program with a

follow-on assignment as an instructor at the United States Military Academy, West Point.

Maria completed all coursework with this final thesis requirement to earn her master's

degree in Chemistry from North Carolina State University, under the direction of Doctor

Russell J. Linderman.

Maria is married to Gregory Miller and has a 13-year old stepson, Greg Jr.

iii

ACKNOWLEDGEMENTS

I owe my greatest debt of gratitude to my advisor, Dr. Linderman. He gave me the

encouragement and guidance I needed to make it through the demanding curriculum and

research. I admire his technical competence and wish him the very best as he moves on

from the world of academia.

I owe many, many thanks to everyone in my group, especially Emmeline Igboekwe

for re-acquainting me with the lab after 12 years. Also, my deepest appreciation to Dr.

Zhengou Zhang, Dr. Matt Isherwood, and Dr. Jaya Prabhakaran for answering all of my

stupid questions and helping me through each and every reaction.

I also thank everyone from Dearstyne's Entomology Department; especially Dr. Roe,

Dr. Deborah Thompson, and Dr. Hugh Young for helping me understand the bug basics

and statistics. And of course, I owe many thanks to Billy D. Chism for the "hand fed"

mosquitoes. That was a new and exciting experience for me. I feel privileged to have

been introduced to the world of bugs. I learned a great deal and had a great time.

I thank Mr. Bob Blinn for the photo shots. I deeply appreciate Rachella Dobson and

Mrs. Norma McDonald for providing a comfortable place to retreat, whenever I had the

chance.

BEST WISHES TO ALL OF YOU!

iv

TABLE OF CONTENTS

page

LIST OF FIGURES������������������������ vi

CHAPTER I

INTRODUCTION AND BACKGROUND������������ 1

AEDES AEGYPTI

History������������������������ 2

Digestion System�������������������� 3

Trypsin Modulation������������������� 4

CHAPTER II

PURPOSE����������������������� � 6

SYNTHESIS OF ANALOGS����������������� 7

CHAPTER III

APPLICATION���������������������� 14

RESULTS������������������������.. 16

CONCLUSIONS���������������������� 18

EXPERIMENTAL PROCEDURES�����������������... 19

LITERATURE CITED���������������������...� 34

v

LIST OF FIGURES

page

Figure 1. Photo of Aedes Aegypti mosquito*��������������� COVER

Figure 2. Distribution of Aedes aegypti in the Americas����������� 2

in 1970 (a) and 1997 (b)

Figure 3. Photo of isolated Aedes aegypti larval midgut ��������..�.... 4

Figure 4. Computer model simulation of TMOF structure�����.���� 4

Figure 5. Photo of Aedes aegypti larvae fed Chorella (outside)�������� 5

and Chorella with TMOF (inside)

Figure 6. Target compound structures����������������� 7

Figure 7. General scheme for retrosynthesis��������������.. 8

Figure 8. Synthetic scheme for target compounds 1f-1h���������� 9

Figure 9. Synthetic scheme for target compounds 2f-2h���������� 11

Figure 10. Synthetic scheme for target compounds 3f-3h���������� 13

Figure 11. Graphs of TMOF vs. analogs (LC50)�������������� 16

Figure 12. Photo of Aedes aegypti larva fed yeast (left) and yeast ������� 17

with target compound (right)

*see endnotes for source

vi

CHAPTER I INTRODUCTION AND BACKGROUND Dengue fever and dengue hemorrhagic fever are mosquito-borne diseases, which even

today, present public health problems in more than 100 countries in Africa, the Americas,

the Eastern Mediterranean, South-East Asia and the Western Pacific.1 These viruses are

transmitted to humans when bitten by infected female Aedes, primarily Aedes aegypti,

mosquitoes. Long ago the yellow fever epidemic was a huge concern, spread solely by

Aedes aegypti.2 Efforts to control the vector have been sought since the turn of the

century. Vast campaigns were used in the 1950s and 1960s to eradicate Aedes aegypti in

tropical and subtropical regions of the Americas.3 Figure 2 indicates the distribution of

Aedes aegypti in the Americas in 1970 (a) at the end of the eradication program, and in

1997 (b) after social and economic change had caused a re-infestation.4 Industrialization

of Latin America, matched with inadequate infrastructure to accommodate increased

urban populations, has undoubtedly caused the re-emergence of Aedes aegypti and the

potential risk of mosquito-borne disease.5

Methods of biological control still remain experimental. Although parasitic

organisms, such as Bacillus thuringiensis (BTI) or larvivorous fish, can be used to

specifically target Aedes aegypti, the expense of producing and difficulty in applying the

organisms limits their effectiveness.6 Current chemical methods of control are less

expensive to produce and easier to use; however, they do not specifically target the

mosquito and pose additional health concerns to other forms of life, including humans.

1

Additionally, Aedes aegypti exhibit resistance to organophosphorus (OP) and dichlor-

diphenyltrichlor (DDT) chemical insecticides that have been used for more than fifteen

years.7

Figure 2. Distribution of Aedes aegypti in the Americas in 1970(a) and 1997(b).

This thesis describes a new chemical approach to target Aedes aegypti mosquitoes,

which can be used safely and effectively, without concerns about stability or cost. Nine

organic compounds have been synthesized and assayed for pesticidal effectiveness

against Aedes aegypti. The synthesis and results are detailed in the following chapters.

AEDES AEGYPTI

History

Aedes aegypti originally came from West Africa and, in the 17th century, spread

throughout the world due to slave trade.8 The organism was transported to the Americas,

by laying eggs in the ships� water cisterns; feeding off the blood of its shipmates.9 By the

time Aedes aegypti landed, it had already adapted to indoor living. 10 Upon arrival, Aedes

2

aegypti began to spread disease; it learned to bite without attracting its host�s attention.11

The mosquito can transmit a virus, just from the action of probing, even without

feeding.12 Now, the mosquito lives outdoors, breeding primarily in man-made

containers.13

Digestive System

In the larval mosquito, food particles are filtered by the pharyngeal fringes, collected

at the back of the pharynx, and gradually swallowed.14 The food passes through the

esophagus to the mid-gut, where it is compacted into a column and enveloped by a

tubular membrane, not touching the gut wall.15 Contractions of the gut wall force the

food out through the rectum as feces.16 It has been shown that it takes 40 minutes for

food to pass through the entire gut, depending on the rate of intake, but regardless of the

size of food particles or nature of the food.17

The blood meal, taken only by female mosquitoes, passes to the posterior midgut (see

Figure 4).18 The most important part of the food requiring digestion is polymeric

material: starch and cellulose, hemicellulose, and proteins.19 Polymer hydrolases are

responsible for breaking down polymeric material; the hydrolase found in the midgut of

Aedes aegypti, of interest, is trypsin.20 This enzyme is key in the digestion of the blood

meal, in egg development, and in survival of the insect.21

3

Figure 3. Photo of isolated Aedes aegypti larval midgut.

Trypsin Modulation

Research done mostly in the past ten years demonstrates that trypsin modulating

oostatic factor (TMOF) inhibited the biosynthesis of trypsin, and therefore blood

digestion and egg development, in the midgut of Aedes aegypti.22 Without trypsin,

Aedes aegypti larvae will not digest food and will die of starvation, even if they continue

eating, otherwise they will not develop due to a lack of sufficient protein.23 TMOF, a

decapeptide with the sequence NH2-Try-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-COOH

(see Figure 5) is an insect hormone that turns off production of trypsin.24

Figure 4. Computer model simulation of TMOF structure.

4

TMOF has been isolated from the ovaries of female Aedes aegypti.25 When

synthesized, by standard solid-phase peptide techniques, and injected into blood-fed

female Aedes aegypti, egg development was significantly inhibited.26 Trypsin assays

also showed a decrease in trypsin activity after TMOF injection. The photo below

(Figure 6) shows the difference in larval development after nine days when fed Chlorella

with TMOF (outer larvae) and fed only Chlorella (inner larvae).27

Figure 5. Photo of Aedes aegypti larvae fed with TMOF (outer) and with out TMOF (inner).

5

CHAPTER II

PURPOSE

Because it is so important to control Aedes aegypti populations, due to the current

transmission of mosquito-borne disease and the likelihood of future worldwide outbreaks,

a safe effective mosquitocide must be developed. Current chemical insecticides cause

undesired side effects and are environmentally unsafe, while biological insecticides are

costly and far less effective.

This research will explore the possibilities of identifying small, organic, non-peptide

compounds, which may exhibit biological activity similar to TMOF. Our initial

investigations are designed to mimic the amino terminus of the decapeptide. Ultimately,

these compounds will be assayed against Aedes aegypti larvae using a feeding assay. The

method used in these feeding assays has incorporated the verification of TMOF activity,

using TMOF as a control.

The synthesis of the initial target compounds and the biological assay method

performed are described herein.

6

SYNTHESIS

Experimental Design

All nine compounds (see Figure 7) were designed to be synthesized in eight steps or

less from three different commercially available starting materials: o-anisaldehyde, m-

anisaldehyde, and 3-(3,4-dimethoxyphenyl)-1-propanol. The retrosynthetic approach is

shown in Figure 8.

Figure 6. Target compound structures. 7

The target compounds are free acids containing an alkene and aryl hydroxy or

methoxy functional groups. The first disconnection should occur at the alkene. After the

alkyl ester is added via a Claisen allyl ether rearrangement, the ester can be hydrolyzed to

form the free acid. This rearrangement was selected in order to get the desired trans

configuration for the alkene. When used with an allylic alcohol, the rearrangement

causes an addition of two carbon atoms and simultaneous repositioning of the double

bond. The allylic alcohol is easily formed via a Grignard addition using vinyl magnesium

bromide with the appropriate dihydrocinnamaldehyde. For target compounds 3f-3h, the

starting material was an alcohol, which was oxidized to the dihydrocinnamaldehyde. For

the other six target compounds, the dihydrocinnamaldehyde was formed by reducing the

ester. These acrylic esters were produced via the Horner-Wadsworth-Emmons

modification of the Wittig reaction from the starting anisaldehydes.28

Figure 7. General scheme for retrosynthesis.

The detailed scheme for each step is shown in the following figures. In the first step,

triethyl phosphonoacetate was deprotonated with a strong base, then the aldehyde is

added. The electrophilic carbon of the carbonyl group was attacked to form the

8

conjugated ester 1a in 97% yield. When NaH was used as the base, the reaction was

complete in 1 hour. This was only attempted once, and a lower yield was obtained.

Then, the double bond is reduced by hydrogenation using palladium on activated

carbon (10 weight %) to form 1b in 91% yield.29 The yields for these steps were very

high and both improved with longer periods of stirring. The DIBAL reduction, however,

always resulted in over-reduction of the ester to a mixture of aldehyde 1c and alcohol

1c'.30 Several reaction conditions were attempted to improve the yield of aldehyde; the

best results occurred when the reaction was stopped after 1 hour. The alcohol was

oxidized using PCC to form 1c; however, those reactions were done separately and not

included in these results.31 When lithium aluminum hydride was used on 1a, a large

mixture of products resulted; therefore, that method was abandoned.32 The Grignard

addition worked quite well yielding 93% of 1d.33

Figure 8. Synthetic scheme for target compounds 1f-1h. 9

The allylic alcohol was heated for 1 hour with triethyl orthoacetate and 3 drops of

propionic acid at 140 ºC. Then the temperature was increased to 170 ºC, and the mixture

refluxed for 7-12 hours.34 The longer it was heated, the better the results. The solvent

was vacuum distilled and the product 1e used without further purification. This ester was

hydrolyzed using potassium hydroxide in methanol and water (5:1).35 The yield was

82%.

Figure 8. Synthetic scheme for target compounds 1f-1h (cont.)

10

The acid 1f was easily separated by acid/base extraction, but still purified by

flash chromotagraphy. Hydrogenation of 1f was not the initial plan. The demethylation

was supposed to come before hydrogenation in the original scheme; however, because

attack of the alkene by HBr was likely occuring and may have caused cyclized by-

products, the procedure was switched. Several attempts were made to deprotect the

hydroxy functional group in all three schemes; however, BBr3 and BCl3 never worked

sufficiently to yield the desired final products. The reactions of the meta-methoxy

substituted compounds paralleled those of the ortho-methoxy substituted compounds.

The yields were very similar for the first two reactions; however, the DIBAL reaction

was significantly improved to yield 66% of the desired aldehyde.

Figure 9. Synthetic scheme for target compounds 2f-2h.

11

The Grignard addition yielded slightly less allylic alcohol and the next two steps yielded

less acid 2g, 78%. It was easy to identify that all of the allylic alcohol had dissappeared

and formation of the acid had occurred by 'H NMR. The alcohol shows two distinct sets

of peaks at 5 and 6 ppm, while the acid shows one large multiplet centered at 5.5 ppm.

Figure 9. Synthetic scheme for target compounds 2f-2h (cont.).

It was not necessary to start with 3,4-dimethoxybenzaldehyde, since 3-(3,4-

dimethoxyphenyl)-1-propanol was available; thereby, eliminating two steps from the

linear synthesis. Undesired side reactions occurred for several trials, both during the

Claisen allyl ether rearrangement and the demethylation, likely due to the increased

activity of the dimethoxy substituted phenyl ring; however, acid 3f was obtained in 70%

yield.

12

Figure 10. Synthetic scheme for target compounds 3f-3h.

13

CHAPTER III: APPLICATION

All feeding study trials were conducted with F3 and F4 generation Aedes aegypti

collected from Raleigh, North Carolina and maintained according to standard procedure

at Dearstyne Entomology Laboratory at North Carolina State University.

Aedes aegypti eggs were hatched from a strip of paper immersed in distilled water

containing dehydrated nutrient broth (DIFCO Laboratories). After 4 hours, the strip was

removed, so that no new hatching occurred; thereby, most larvae would be of the same

instar. After 24 hours, individual larvae were transferred into the wells of a 96 well

microtiter plate (flat-bottom, non-sterile) along with 60 µL of distilled water using an

automatic pipette.

To each well, 10 µL of 2% brewer's yeast (debittered, inactive) in distilled water

and enough additional distilled water to bring the total volume per well to 188 µL was

added. For the TMOF controls, a solution of 20 mg/mL distilled water was prepared

for use. Three rows of 12 mosquitoes were treated with a concentration of TMOF

solution: 2 mg/mL (18 µL of 20 mg/mL TMOF solution), 1 mg/mL (9 µL of solution),

and 0.5 mg/mL (4.5 µL of solution). A total of 108 larvae were treated; therefore, more

than one microtiter plate was needed per trial.

For each analog, a total of 176 µL of distilled water, 10 µL of 2% brewer's yeast

(debittered, inactive) in distilled water, and 2 µL of compound dissolved in dimethyl

sulfoxide (DMSO) were added to each well. The analog concentrations were prepared by

14

making two-fold dilutions into DMSO from the initial concentration (about 0.8 mM,

0.4 mM, and 0.2 mM).

Blanks were also prepared by adding 178 µL of distilled water and 10 µL of 2%

brewer's yeast to each of 36 wells; and, adding 176 µL of distilled water, 10 µL of 2%

brewer's yeast, and 2 µL of DMSO to each of 36 wells.

The larvae were reared in a climate chamber maintained at 26 ºC with a 16:8 hour

light:dark cycle and larvae were checked for mortality every 12 hours over a period of

five days, using a dissecting microscope. The microtiter plates were kept in a closed

container with damp paper towels, in order to keep evaporation at a minimum. Figure 12

shows an enlarged view of the first two instars. Only first and second instars were

treated.

It was quite difficult to determine whether the larvae were still alive. It was helpful to

use a direct beam of light, which causes them to become agitated. If they did not move

when light was directed into their well nor after tapping the microtiter plate gently on the

base of the microscope, then they were not alive. Also, the dead larvae usually were fully

extended and more translucent. After three days, the water sometimes became cloudy

(probably due to the yeast), which made it difficult to see the larvae.

15

RESULTS Six of the nine compounds of the structures shown in Figure 7 were synthesized in

seven steps or less. Following the procedure in the previous section, three replicates of

twelve larvae were used in a feeding assay with three different concentrations of each

analog and TMOF, as well as blanks. The LC50 was determined using the percent larvae

killed at each concentration (see Appendix A). These compounds, originally designed as

analogs of the decapeptide TMOF (trypsin modulating oostatic factor), were far more

effective against Aedes aegypti. These compounds responded faster and at lower

concentrations than TMOF. The graph in Figure 13 shows the LC50 for each analog

compared to TMOF. LC50, is the concentration of larvicide required to be lethal to 50%

of the target population. Because all of the tested analogs have a lower LC50 than TMOF,

they are all more effective. And, these compounds are far less costly to produce.

Figure 11. LC50 plot (TMOF vs. analog)

16

Figure 11. LC50 plot (TMOF vs. analog) (cont.)

The photos below (Figure 12) show results of feeding a TMOF analog 7-(3,4-

dimethoxyphenyl)-4-heptenoic acid (3f) to Aedes aegypti. The mosquito larva on the left

was fed only yeast. After four days it is still alive with molted head casing floating in the

well. The larva on the right was fed 0.7mM concentration of the analog and died within

12 hours. The photograph of the treated larva is magnified compared to the untreated

larva. The treated larva did not appear to successfully molt to the next stage.

17

CONCLUSIONS Six of nine proposed compounds were synthesized and used in feeding studies with

Aedes aegypti. The compounds were designed as analogs of trypsin modulating oostatic

factor (TMOF). TMOF is effective against this and other species of mosquitoes through

regulation of the digestive enzyme trypsin. The synthetic compounds were even more

effective than TMOF against Aedes aegypti. This may not be due to trypsin modulation,

since they work so quickly. They may merely be toxins absorbed by the larvae. In either

case, the compounds do exhibit the larvacidal activity as observed for TMOF and would

be a less costly, more effective alternative.

18

EXPERIMENTAL PROCEDURES

General

All reagents were purchased from Aldrich, Fisher Scientific, or Sigma unless

otherwise noted. All reagents were purified prior to use, when necessary.

Tetrahydrofuran was distilled from sodium-benzophenone and methylene chloride from

CaH2. All reactions were done under argon in oven-dried glassware, unless otherwise

noted.

Fourier transform infrared spectra were recorded on a Perkin-Elmer 1600 series

spectrophotometer. Nuclear magnetic resonance spectra, proton and carbon, were

recorded on a General Electric or Varian 300 MHz spectrometer using CDCl3 as the

solvent. Chromatography was performed on silica gel 60, 230-400 mesh ASTM from EM

Science. Whatman thin layer chromatography (TLC) plates having a coating of

fluorescent silica gel on aluminum backing were used for TLC analyses. Visualization of

TLC plates was possible by ultraviolet light and 5% solution of phosphomolybdic acid in

ethanol.

19

General Procedures

Horner-Wadsworth-Emmons Reaction. To a mixture of lithium hydroxide hydrate

(1.1 eq) in THF (45 mL) was added triethyl phosphonoacetate (1.1 eq). After stirring

under argon for ten minutes, commercially available aldehyde (1 eq) was added and

allowed to stir at room temperature for 48 hours. The mixture was extracted with ethyl

ether (anhydrous) and the organic layer dried with magnesium sulfate. The solvent was

removed under vacuum.

Hydrogenation. Ethanol (45 mL) was added to the round-bottom flasks containing the

conjugated esters. The flask was flushed with argon before adding palladium on

activated carbon (10 wt %). The argon was removed from the flask and a balloon filled

with hydrogen was attached. The mixture was stirred for 20 hours. The mixture was

filtered through celite and the solvent removed under vacuum. The same procedure was

used to reduce the alkene of the acid targets 1f-3f.

Diisobutylaluminum Hydride (DIBAL) Reduction. The esters were stirred in methylene

chloride (20 mL) and cooled to -78 oC (CO2/acetone). DIBAL (1.1 eq) was added to the

mixture and allowed to stir for 1 hour at -78 oC. The reaction was quenched with

aqueous sodium potassium tartrate, filtered through celite, and extracted with methylene

chloride. The organic layer was dried with magnesium sulfate and the solvent removed

under vacuum.

20

Pyridinium Chlorochromate (PCC) Oxidation. PCC (1.5 eq)/celite and methylene

chloride (20 mL) was stirred together with 4Å molecular sieves for 10 minutes at room

temperature. The alcohol (1 eq) was added to the mixture and allowed to stir for 2 hours.

The mixture was filtered through celite and forced through a column of silica gel to

eliminate residue.

Grignard Addition. The aldehyde (1 eq) in tetrahydrofuran (40 mL) was added to vinyl

magnesium bromide (1.5 eq) under argon at 0 oC . The solution was stirred and allowed

to warm to room temperature over 2 hours. The ice bath was replaced and aqueous

ammonium chloride was added. The mixture was extracted with ethyl ether. The organic

layer was washed with aqueous sodium bicarbonate and brine, dried over magnesium

sulfate, and the solvent removed under vacuum.

Claisen Allyl Ether Rearrangement. Triethyl orthoacetate (10 eq) was added to the

alcohols along with propionic acid (cat.) and heated to 140 oC for 1 hour. The

temperature was increased to 170 oC for an additional 7 hours. The solvent was distilled

off under reduced pressure at 60 oC. The crude material was used for the next step

without purification.

Saponification. The crude esters were stirred for 4 hours with potassium hydroxide (3

eq) and a mixture of methanol (5 mL) and distilled water (1 mL). The solvent was

21

removed under vacuum and the residue was partitioned between water and ethyl ether.

The water layer was acidified with 6N hydrochloric acid to pH 2 at 0 o C and extracted

with ethyl ether. The ether layer was washed with water and brine, and dried over

sodium sulfate. The solvent was removed under vacuum to yield a pale yellow oil.

Demethylation. The ester (1 eq) was dissolved in methylene chloride (20 mL) and cooled

to �78 oC (CO2/acetone). Boron tribromide (3 eq) was added and the mixture was stirred

for 2 hours. The reaction was quenched with water. The mixture was extracted with

methylene chloride and the organic layer dried with sodium sulfate. The solvent was

removed under vacuum.

22

Experimental Results

3-(2-Methoxyphenyl)-2-propenoate (1a). o-Anisaldehyde (2.000 g, 14.690 mmol),

lithium hydroxide hydrate (0.678 g, 16.159 mmol), triethyl phosphonoacetate (3.20 mL,

16.159 mmol) were used in the Horner-Wadsworth-Emmons reaction. The oily crude

product was flash chromatographed on a silica gel column and eluted with 2% ethyl

acetate in hexane to yield 97% (2.939 g) of 1a. IR (neat): 3412, 2985, 2846, 1713, 1632,

1600, 1467, 1317, 1162, 1029, 869 cm-1. 1H NMR (300 MHz, CDCl3): 7.95 (d, 1 H,

J = 16 Hz), 7.46 (t, 1 H, J = 7.5 Hz), 7.28 (t, 1 H, J = 8 Hz), 6.87 (m, 2 H), 6.48 (d, 1 H,

J = 16 Hz), 4.21 (q, 2 H, J = 7 Hz), 3.81 (s, 3 H), 1.28 (t, 3 H, J = 7 Hz) δδδδ. 13C NMR (300

MHz, CDCl3): 14.36, 55. 41, 60.30, 111.11, 118.76, 120.66, 128.87, 131.41, 139.97 δδδδ.

3-(3-Methoxyphenyl)-2-propenoate (2a). m-Anisaldehyde (2.000 g, 14.690 mmol),

lithium hydroxide hydrate (0.678 g, 16.159 mmol), triethyl phosphonoacetate (3.20 mL,

23

16.159 mmol) were used in the Horner-Wadsworth-Emmons reaction. The oily crude

product was flash chromatographed on a silica gel column and eluted with 2% ethyl

acetate in hexane to yield 98% (2.969 g) of 2a. IR (neat): 2985, 2836, 1712, 1604, 1513,

1167, 1031, 828 cm-1. 1H NMR (300 MHz, CDCl3): 7.64 (d, 1 H, J = 16 Hz), 7.29 (t,

1 H, J = 8 Hz), 7.11 (d, 1 H, J = 7.5 Hz), 7.03 (s, 1 H), 6.92 (m, 1 H), 6.42 (d, 1 H,

J = 16 Hz), 4.26 (q, 2 H, J = 7 Hz), 3.82 (s, 3 H), 1.33 (t, 3 H, J = 7 Hz) δ. δ. δ. δ. 13C NMR (300

MHz, CDCl3): 14.36, 55.31, 60.55, 112.92, 116.13, 118.60, 120.78, 129.89, 144.52 δ.δ.δ.δ.

3-(2-Methoxyphenyl)propanoate (1b). Hydrogenation procedure was carried out on 1a

(2.939 g, 14.250 mmol) forming a yellow oily substance that was purified by flash

chromatography on a silica gel column and eluted with 100% hexane to yield 91%

(2.701 g) of 1b. IR (neat): 2921, 2857, 1740, 1649, 1558, 1542, 1451, 1034 cm-1. 1H

NMR (300 MHz, CDCl3): 7.15 (m, 2 H), 6.85 (m, 2 H), 4.12 (q, 2 H, J = 7 Hz), 3.83 (s,

3 H), 2.94 (t, 2 H, J = 8 Hz), 2.60 (t, 2 H, J = 8 Hz), 1.24 (t, 3 H, J = 7 Hz) δ. δ. δ. δ. 13C NMR

(300 MHz, CDCl3): 14.26, 26.15, 34.28, 55.20, 60.28, 110.22, 120.42, 127.57, 129.97 δ.δ.δ.δ.

24

3-(3-Methoxyphenyl)propanoate (2b). Hydrogenation procedure was carried out on 2a

(2.969 g, 14.396 mmol) forming a yellow oily substance that was purified by flash

chromatography on a silica gel column and eluted with 100% hexane to yield 89%

(2.668 g) of 2b. IR (neat): 2932, 2836, 1734, 1600, 1595, 1494, 1456, 1371, 1258, 1152,

1045, 692 cm-1. 1H NMR (300 MHz, CDCl3): 7.20 (m, 1 H), 6.78 (m, 3 H), 4.13 (q,

2 H,J = 7 Hz), 3.79 (s, 3 H), 2.93 (t, 2 H, J = 8 Hz), 2.62 (t, 2 H, J = 8 Hz), 1.24 (t, 3 H,

J = 7 Hz) δ. δ. δ. δ. 13C NMR (300 MHz, CDCl3): 14.26, 26.15, 34.28, 55.20, 60.28, 110.22,

120.42, 127.57, 129.97 δ.δ.δ.δ.

3-(2-Methoxyphenyl)propanal (1c). DIBAL (14.26 mL, 1.0 M in DCM) was added to

1b (2.701 g, 12.968 mmol) and reduction procedure followed. The resulting crude yellow

oil was further purified by flash chromatography (silica gel), eluting with 5% ethyl

acetate in hexane to yield 56% (1.192 g) of 1c and 31% (0.732 g) of 1c'. IR (neat):

25

2935, 2835, 2726, 1725, 1600, 1496, 1466, 1247, 1117, 1038, 754 cm-1. 1H NMR (300

MHz, CDCl3): 9.77 (s, 1 H), 7.16 (m, 2 H), 6.87 (m, 2 H), 3.80 (s, 2 H), 2.94 (t, 2 H, J =

6 Hz), 2.69 (t, 2 H, J = 6 Hz) δ. δ. δ. δ. 13C NMR (300 MHz, CDCl3): 23.53, 43.85, 55.15,

110.30, 120.56, 127.74, 129.96 δ.δ.δ.δ.

3-(3-Methoxyphenyl)propanal (2c). DIBAL (14.09 mL, 1.0 M in DCM) was added to

2b (2.668 g, 12.812 mmol) and reduction procedure followed. The resulting crude

yellow oil that was further purified by flash chromatography (silica gel), eluting with 5%

ethyl acetate in hexane to yield 66% (1.388 g) of 2c an 18% (0.420 g) of 2c'. IR (neat):

2921, 2846, 2718, 1718, 1654, 1579, 1456, 1258, 1152, 1034, 858 cm-1. 1H NMR (300

MHz, CDCl3): 9.83 (s, 1 H), 7.22 (m, 2 H), 6.78 (m, 2 H), 3.81 (s, 3 H), 2.95 (t, 2 H,

J = 7), 2.79 (t, 2 H, J=7) δ. δ. δ. δ. 13C NMR (300 MHz, CDCl3): 28.20, 45.18, 55.18, 111.57,

114.20, 120.63, 129.62 δ.δ.δ.δ.

26

3-(3,4-Dimethoxyphenyl)propanal (3c). PCC (3.295g, 15.287 mmol) was added to

3-(3,4-dimethoxyphenyl)-1-propanol (2.000 g, 10.191 mmol) and oxidation procedure

followed to yield 70% (2.089 g) of 3c. IR (neat): 2932, 2846, 2729, 1723, 1504, 1446,

1258, 1146, 1029, 804 cm-1. 1H NMR (300 MHz, CDCl3): 9.82 (s, 1 H), 6.76 (m, 3 H),

3.87 (s, 3 H), 3.85 (s, 3 H), 2.92 (t, 2 H, J = 7 Hz), 2.77 (t, 2 H, J = 7 Hz) δ. δ. δ. δ. 13C NMR

(300 MHz, CDCl3): 14.26, 26.15, 34.28, 55.20, 60.28, 110.22, 120.42, 127.57, 129.97 δ.δ.δ.δ.

5-(2-Methoxyphenyl)-3-hydroxy-1-pentene (1d). The procedure for Grignard addition

was followed with vinylmagnesium bromide (10.89 mL, 1.0 M in THF) and 1c (1.192 g,

7.259 mmol) to yield a yellow oil. The crude product was purified by flash

chromatography on a silica gel column eluted with 25% ethyl acetate in hexane to yield

93% (1.298 g) of 1d. IR (thin film): 3850, 3744, 3391, 2932, 1697, 1643, 1494, 1462,

27

1242, 1040, 917, 746 cm-1. 1H NMR (300 MHz, CDCl3): 7.20 (m, 2 H), 6.89 (m, 2 H),

5.95 (m, 1 H), 5.26 (d, 1 H, J = 17 Hz), 5.12 (d, 1 H, J = 10 Hz), 4.11 (m, 1 H), 3.86 (s,

3 H), 2.74 (m, 2 H), 1.83 (m, 2 H) δ. δ. δ. δ. 13C NMR (300 MHz, CDCl3): 25.93, 37.38, 55.37,

72.13, 110.36, 114.42, 120.71, 127.23, 130.12 δ.δ.δ.δ.

5-(3-Methoxyphenyl)-3-hydroxy-1-pentene (2d). The procedure for Grignard addition

was followed with vinylmagnesium bromide (12.68 mL, 1.0 M in THF) and 2c (1.388 g,

8.453 mmol) to yield 93% (1.511 g) of 2d. IR (thin film): 3425, 2943, 2837, 2743, 1678,

1590, 1449, 1372, 1267, 1167, 1044, 861, 785, 685 cm-1. 1H NMR (300 MHz, CDCl3):

7.29 (m, 1 H), 6.85 (m, 3 H), 5.99 (m, 1 H), 5.33 (d, 1 H, J = 17 Hz), 5.22 (d, 1 H, J = 11

Hz), 4.15 (m, 1 H), 3.89 (s, 3 H), 3.87 (s, 3 H), 2.69 (m, 2 H), 1.86 (m, 2 H) δ. δ. δ. δ. 13C NMR

(300 MHz, CDCl3): 31.72, 38.41, 55.17, 72.50, 111.21, 114.26, 114.98, 120.89, 129.39 δ.δ.δ.δ.

28

5-(3,4-Dimethoxyphenyl)-3-hydroxy-1-pentene (3d). The procedure for Grignard

addition was followed with vinylmagnesium bromide (16.05 mL, 1.0 M in THF) and 3c

(2.089 g, 10.701 mmol) to yield 83% (1.983 g) of 3d. IR (thin film): 3394, 2921, 2858,

1592, 1516, 1456, 1260, 1156, 1028 cm-1. 1H NMR (300 MHz, CDCl3): 6.79 (m, 3 H),

5.93 (m, 1 H), 5.27 (d, 1 H, J = 17 Hz), 5.16 (d, 1 H, J = 10 Hz), 4.15 (m, 1 H), 3.89 (s, 3

H), 3.87 (s, 3 H), 2.69 (m, 2 H), 1.86 (m, 2 H) δ. δ. δ. δ. 13C NMR (300 MHz, CDCl3): 31.60,

38.31, 55.17, 60.57, 72.79, 110.41, 120.89, 127.39 δ.δ.δ.δ.

7-(2-Methoxyphenyl)-4-heptenoic acid (1f). Procedures for the Claisen allyl ether

rearrangement were followed by the ester saponification. Triethyl orthoacetate (7.19 mL,

39.322 mmol) was refluxed with propionic acid (3 drops) and 1d (0.756 g, 3.932 mmol).

After solvent removal, potassium hydroxide (0.662 g, 11.796 mmol) was added. The

yield was 72% (0.663 g) for 1f. IR (thin film): 3354, 2919, 2849, 1707, 1494, 1462,

29

1248, 751 cm-1. 1H NMR (300 MHz, CDCl3): 7.17 (m, 1 H), 7.12 (m, 1 H), 6.86 (m,

2 H), 5.51 (m, 2 H), 3.82 (s, 3 H), 2.66 (t, 2 H, J = 7), 2.39 (m, 2 H), 2.30 (m, 4 H) δ. δ. δ. δ. 13C

NMR (300 MHz, CDCl3): 26.51, 34.28, 34.50, 35.17, 55.20, 110.44, 112.22, 120.41,

127.57, 130.01 δ.δ.δ.δ.

7-(3-Methoxyphenyl)-4-heptenoic acid (2f). Procedures for the Claisen allyl ether

rearrangement were followed by the ester saponification. Triethyl orthoacetate (6.39 mL,

34.953 mmol) was refluxed with propionic acid (3 drops) and 2d (0.672 g, 3.495 mmol).

After solvent removal, potassium hydroxide (0.588 g, 10.489 mmol) was added. The

yield was 78% (0.639 g) for 2f. . IR (thin film): 3355, 2914, 2853, 1707, 1498, 1452,

1250, 756 cm-1. 1H NMR (300 MHz, CDCl3): 7.21 (m, 1 H), 6.77 (m, 3 H), 5.51 (m,

2 H), 3.82 (s, 3 H), 2.66 (t, 2 H, J = 7.5), 2.38 (m, 6 H) δ. δ. δ. δ. 13C NMR (300 MHz, CDCl3):

26.51, 34.28, 34.50, 35.17, 55.23, 111.15, 112.22, 114.41, 120.57, 128.97 δ.δ.δ.δ.

30

7-(3,4-Dimethoxyphenyl)-4-heptenoic acid (3f). Procedures for the Claisen allyl ether

rearrangement were followed by the ester saponification. Triethyl orthoacetate

(7.085 mL, 38.739 mmol) was refluxed with propionic acid (3 drops) and 3d (0.865 g,

3.874 mmol). After solvent removal, potassium hydroxide (0.652 g, 11.622 mmol) was

added. The yield was 70% (0.717) for 3f. IR (thin film): 3425, 2931, 1713, 1649, 1519,

1455, 1420, 1261, 1237, 1155, 10.26, 803 cm-1. 1H NMR (300 MHz, CDCl3): 6.55 (m, 3

H), 5.50 (m, 2 H), 3.81 (s, 3H), 3.80 (s, 3 H), 2.53 (m, 2 H), 2.35 (m, 6 H) δ. δ. δ. δ. 13C NMR

(300 MHz, CDCl3): 26.51, 34.28, 34.50, 35.17, 55.20, 55.22, 112.22, 120.41, 127.57,

131.97 δ.δ.δ.δ.

7-(2-Methoxyphenyl)heptanoic acid (1g). Hydrogenation of 1f (0.328 g, 1.400mmol)

with palladium on activated carbon (0.033 g, 10 wt %) yielded crude product, which was

31

purified on a silica gel column by flash chromatography, eluted with 20% ethyl acetate in

hexane, to yield 92% (0.300 g) of 1g. IR (thin film): 3354, 2919, 2853, 1708, 1494,

1465, 1248 cm-1. 1H NMR (300 MHz, CDCl3): 7.15 (m, 2 H), 6.87 (m, 2 H), 3.84 (s,

3 H), 2.62 (t, 2 H, J = 7), 2.37 (m, 2 H), 1.39 (m, 8 H) δ. δ. δ. δ. 13C NMR (300 MHz, CDCl3):

24.60, 28.65, 31.10, 33.50, 36.17, 55.83, 110.34, 111.89, 114.26, 120.15 δδδδ.

7-(3-Methoxyphenyl)heptanoic acid (2g). Hydrogenation of 2f (0.417 g, 1.780 mmol)

with palladium on activated carbon (0.042 g, 10 wt %) yielded crude product, which was

purified on a silica gel column by flash chromatography, eluted with 20% ethyl acetate in

hexane, to yield 94% (0.395 g) of 1g. IR (thin film): 3356, 2914, 2853, 1706, 1502,

1453, 1243 cm-1. 1H NMR (300 MHz, CDCl3): 7.15 (m, 2 H), 6.87 (m, 2 H), 3.81 (s,

3 H), 2.66 (t, 2 H, J = 7), 2.37 (m, 2 H), 1.41 (m, 8 H) δ. δ. δ. δ. 13C NMR (300 MHz, CDCl3):

24.60, 28.65, 31.10, 33.50, 36.17, 55.83, 111.25, 114.89, 114.99, 120.75 δδδδ.

32

7-(3,4-Dimethoxyphenyl)heptanoic acid (3g). Hydrogenation of 3f (0.220 g, 0.829

mmol) with palladium on activated carbon (0.022 g, 10 wt %) yielded crude product,

which was purified on a silica gel column by flash chromatography, eluted with 20%

ethyl acetate in hexane, to yield 86% (0.190 g) of 3g. IR (thin film): 3425, 2929, 1715,

1519, 1450, 1421, 1261, 1240, 1145, 1026 cm-1. 1H NMR (300 MHz, CDCl3): 6.77 (3

H), 3.90 (s, 3 H), 3.88 (3 H), 2.57 (t, 2 H, J = 7.5), 2.37 (t, 2 H, J = 7), 1.41 (m,

8 H) δ. δ. δ. δ. 13C NMR (300 MHz, CDCl3): 24.63, 28.89, 31.46, 33.82, 35.47, 55.83, 55.94,

111.24, 111.79, 120.15 δδδδ.

33

LITERATURE CITED

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(b) Taubes, G. Tales of a Bloodsucker. Discover Magazine 1998, July. (c)

Henderson, A. D.; Vector Control. Malaria Weekly 1996, 12.

2. Ibid.

3. (a) http://www.cdc.gov/ncidod/dvbid/dengue.htm. (b) Potential Risk for Dengue

Hemorrhagic Fever: The Isolation of Serotype Dengue-3 in Mexico. Emerging

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4. Ibid.

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6. http://www.who.org/inf-fs/en/fact117.html

34

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8. (a) Christophers, S. R. Aedes aegypti (L.), The Yellow Fever Mosquito: its Life

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1960. (b) Taubes, G. Tales of a Bloodsucker. Discover Magazine 1998, July.

9. Ibid.

10 Ibid.

11. Ibid.

12. Taubes, G. Tales of a Bloodsucker. Discover Magazine 1998, July.

13. (a) http://www-personal.usyd.edu.au/~sdoccett/fact/dengue.htm. (b)

http://www.who.org/inf-fs/en/fact117.html

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15. Ibid.

16. Ibid.

17. Ibid.

18. Terra, W. A. Evolution of Digestive Systems of Insects. Annual Reviews in

Entomology 1990, 35, 181-200.

19. Ibid.

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21. Ibid.

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23. http://www.insectbio.com/tech.htm

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37

Biochemical and Biophysical Research Communications 1993, 193, 688-693. (b)

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39

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*http://klab.agsci.colostate.edu/aegypti/aegypti.html

40