natural products as repellents and insecticides – charles cantrell

United States Department of Agriculture – Agricultural Research Service

Natural Products as Repellents and Insecticides – Charles Cantrell

•Callicarpenal Update•DEET and Picaridin Analogs

•(Conclusion of Large SAR Study published in Chemistry and Biodiversity)

•Callicarpenal Extraction Study •(Needed for Scale-up of Callicarpenal for commercial partners)

•Inula helenium Analogs •Amine Analogs•(Native plant to MS with mosquito actives)


C. americana Compounds at 25 nmole/cm2 vs. Aedes aegypti and Anopheles stephensi

CHO

H N

O


Callicarpenal Michael Addition Products

H

O

N

CHO

H

1

4

9

1220

17

19

18

COOH

H

CrO3, H2SO4

acetone, H2O

DCM, Et3N

H

O

N

ClCO2Et

HN

DCM, Et3N

ClCO2Et

HN

Overall 22 % yield

Overall 27 % yield

Intermediate not isolated

N

O

N

OO*

*CH2CH2OH

DEETPicaridin


Callicarpenal Michael Addition Products

H

O

N

H

O

NCHO

H


Callicarpenal Sourcing Possibilities

•Callicarpa americana•Callicarpa sp.•Verbenaceae species•Methods•Time of Year

•Smaller cpd. build-up•Larger cpd. breakdown

•Wieland-Miescher•Sclereolide•Labdane to Clerodane

QSAR

Simple/Cheap Analog


Purity analysis of callicarpenal in hexane, methylene chloride (DCM), methanol, and steam distillation extracts


Seasonal variation of callicarpenal collected from two separate locations in Mississippi


Natural Products as Repellents and Insecticides

•Callicarpenal Update•DEET and Picaridin Analogs•Extraction Study•Inula helenium Analogs •Amine Analogs

Day 1: Hatch Aedes aegypti eggs under vacuum

Day 2: Sort 1st instar larvae to 24-well plates, 5/well; Add 1ml of water and food; Add 10 ul of insecticide solution

Day 3: Score bioassay data (dead/total)

High throughput larvacidal screen – Dr. Becnel’s Group


,-unsaturated lactone reactivity

O

O

HNu

O

O

H

Nu

H

O

O

HNu

RSH


CPD ID Reactant Nucleophile 125 ppm

62.5 ppm

31.25 ppm

15.625 ppm

8 ppm

4 ppm

CLC11062A Isoalantolactone nonyl amine 100 33.3 20 6.67 0 0

CLC11063B alantolactonenonyl amine 100 100 53. 13.3 0 0

CLC11064A Isoalantolactone pyrrolidine 20 6.67 0 0 0 0

CLC11065B alantolactone pyrrolidine 100 93.3 20 0 0 0

CLC11066A isoalantolactone benzyl amine 0 0 0 0 0 0

CLC11067A alantolactonebenzyl amine

53.3 33.3 6.6 0 0 0

CLC11068A isoalantolactone 3-chloro-N-methylbenzylamine 0 0 0 0 0 0

CLC11069A alantolactone3-chloro-N-

methylbenzylamine 6.67 0 0 0 0 0

CLC11070B isoalantolactone 4-mercaptopyridine 13.3 6.67 0 0 0 0

CLC11071A alantolactone 4-mercaptopyridine 40 26.7 20 13.3 6.67 0

HN

H2N

H3CHNCl

NHS

Percent mortality of isoalantolactone and alantolactone isomers against A. aegypti larvae

H2N (CH2)8 CH3


Alantolactone (1a) and isoalantolactone (2a) Micheal addition reaction products.

O

O

H

O

O

3a R = diethyl amine CLC11058A3b R = piperidine CLC11060A3c R = nonyl amine CLC1163B3d R = pyrrolodine CLC1165B3e R = benzyl amine CLC1167A3f R = 3-chloro-N-methylbenzyl amine CLC1169A3g R = 4-mercaptopyridine CLC1171A3h R = propyl amine CLC11073B3i R = hexyl amine CLC11076A3j R = butyl amine CLC11078A3k R = decyl amine CLC11080A3l R = undecyl amine CLC11082A3m R = dodecyl amine CLC11084A

4a R = diethyl amine CLC11055A4b R = piperidine CLC11059A4c R = nonyl amine CLC1162A4d R = pyrrolodine CLC1164A4e R = benzyl amine CLC1166A4f R = 3-chloro-N-methylbenzyl amine CLC1168A4g R = 4-mercaptopyridine CLC1170B4h R = propyl amine CLC11074A4i R = hexyl amine CLC11075A4j R = butyl amine CLC11077A4k R = decyl amine CLC11079A4l R = undecyl amine CLC11081A4m R = dodecyl amine CLC11083A

R R


Four to twelve carbon aliphatic Michael addition substituted reaction products versus larval LC50 activity. Corresponding second order polynomial regression lines are overlaid.

O

O

HR


Four to twelve carbon aliphatic Michael addition substituted reaction products versus larval LC50 activity. Corresponding second order polynomial regression lines are overlaid.

O

O

R


2010 Research Plans

Additional Michael addition analogs of Inula sp. compounds.

Intermedeol SAR study. Work with commercial partner towards

commercialization of callicarpenal.


•330 plant extracts •118 pure compounds

have been submitted for Larvicidal and Adult Topical assays.

•97 plant extracts •32 pure compounds

have been submitted for Repellent assay.

Work of David Wedge and Nur Tabanca


Aedes aegypti 1st instar Larval BioassayDead out of a total of 5 larvaeDead out of a total of 5 larvae

181 essential oils have been submitted so far.The most active ones:

Requested scale up for more in depth studies

Species 31.25 ppm

15.625 ppm

8 ppm

Ledum palustre 5 5 3 Aegopodium alpestre 5 3 1 Dracocephalum moldavicum 5 3 3 Paeonia anomalia 4 4 2

Plant essential oils from Turkish collaborators


0

20

40

60

80

100

120

8 ppm 4 ppm 2 ppm 1 ppm

24h-

mor

talit

y (%

)

E-1

E-2

E-3

E-4

High throughput larval bioassay41 Euphorbiaceae extracts were screened against Ae. Aegypti.

Recently started fractionation


Aedes aegypti 1st instar Larval Bioassay

Dead out of a total of 5 larvaeDead out of a total of 5 larvae

JWN# 31.25 ppm

15.625 ppm

8 ppm

4 ppm

319 5 5 4 4 330 5 3 4 1 417 5 4 4 0 419 5 5 5 5 628 5 5 3 0 683 5 5 5 0

Of the 118 pure compounds

*

* initiated more in depth studies

*


Aegypti Minimum Effective Dosage (mg/cm²)

0.000

0.020

0.040

0.060

0.080

0.100

0.120

0.140

0.160

0.180

0.200

DEET

Wedge 1-1

Wedge 1-2

Wedge 1-5

Wedge 1-6

Wedge 1-8

Wedge 1-7

Wedge 1-9

Samples

Dos

age

(mg/

cm²)

Mosquito repellent activity of crude plant extracts


Aegypti Minimum Effective Dosage (mg/cm²)

0.000

0.050

0.100

0.150

0.200

0.250

0.300

DEET

Wedge 2-12

Wedge 2-1

Wedge 2-2

Wedge 2-4

Wedge 2-11

Wedge 2-13

Wedge 2-15

Wedge 2-9

Wedge 2-10

Wedge 2-14

Wedge 2-8

Wedge 2-7

Wedge 2-5

Wedge 2-3

Wedge 2-6

Samples

Dosa

ge (m

g/cm

²)Mosquito repellent activity of pure compounds (*)

and crude plant extracts

* * * * * * **

*

* *


Future work• Bioassay-guided fractionation will be performed on most active extracts to identify active compounds

• 29 NEW aromatic species and will be examined for repellent and insecticidal activity

•20 fungal endophyte extracts from Brazil will be submitted after preliminary studies are complete

• We anticipate several papers, invention disclosures to be sought on most actives with CMAVE, and potential patents

natural products as repellents and insecticides – charles cantrell

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