DITERPENES FROM EUROPEAN EUPHORBIA SPECIES SERVING AS PROTOTYPES FOR NATURAL-PRODUCT-BASED DRUG DISCOVERY

Judit Hohmann Institute of Pharmacognosy, University of Szeged

University of Szeged, Faculty of University of Szeged, Faculty of PharmacyPharmacy

University of Szeged

Faculty of Agriculture

Faculty of Arts

Faculty of Dentistry

Faculty of Economics and Business Administration

Faculty of Engeneering

Faculty of Health and Social Studies

Faculty of Law

Faculty of Medicine

Faculty of Music

Faculty of Science and Informatics

Juhász Gyula Teacher Training Faculty

Faculty of PharmacyFaculty of Pharmacy- Institute of Pharmaceutical Analysis- Institute of Clinical Pharmacy- Institute of Pharmaceutical Chemistry- Institute of Pharmaceutical Technology- Institute of Pharmacodynamics and Biopharmacy- Institute of Drug Regulatory Affairs- Institute of Pharmacognosy

Institute of PharmacognosyInstitute of Pharmacognosy

• Education on five faculties Education on five faculties (Pharmacy, Medicine, Health Social Studies, Agriculture, Teacher Training) Pharmacognosy theory and practice Phytotherapy Elective courses: Separation techniques, Biotechnology, Chemotaxonomy PhD education in Graduate School of Pharmaceutical Sciences

• Research activity aimsResearch activity aims Isolation and structure determination of natural compounds, Preparative scale purification of plant constituents for pharmacological,

analytical studies Standardization, quantitative and qualitative analysis of drugs and herbal

medicinal products, quality control of products and dietary supplements Chemotaxonomy, production biology, studies on the dynamics

of accumulation of bioactive substances in plants

Euphorbiaceae familyEuphorbiaceae family

• Euphorbiaceae family:Euphorbiaceae family:– around 7.500 species in 300 genera– morphology: non-succulent, succulent or cactiform– herbs, shrubs or trees

• Widely used EuphorbiaceaeWidely used Euphorbiaceae species:species:– Castor oil (Ricinus communis)– Rubber (Hevea brasiliensis and other species)– Manihot or cassava starch (Manihot esculenta)– Kamala (Mallotus philippinensis)– Croton oil (Croton tiglium)– Euphorbium (Euphorbia resinifera)– Other 150 species used in the folk medicine against

cancer*

* HartwellJL.Lloydia 1969, 32, 153-205

EuphorbiaEuphorbia genera genera• Number of species, characteristicsNumber of species, characteristics

– one of the largest and most diverse genera in the plant kingdom with ~2000 species

– irritant milky latex content – 105 species in Europe (Tutin et al. Flora Europaea 1968)– 36 species in the Carpathian basin (Jávorka-Csapodi,

Iconographia 1975)– 24 species in Hungary (Simon T. 1992)

• Chemistry Chemistry – Diterpenes: „lower” and „higher” terpenes (from ~200 species)

– Triterpenes (dammarane, lupane, oleanane, cycloartane, seco-derivatives), steroids

– Phenolics, flavonoids, tannins, coumarins– Cerebroside, glycerols– Others: sesquiterpene, etc.

Biogenetic origin of diterpenesBiogenetic origin of diterpenes„Lower terpenes”

Macrocyclic diterpenes and their cyclisation products

„Higher terpenes” Cyclic diterpenes

OPP

GGPP

lathyrane

cembrene cation

jatrophane

ingenane daphnane tigliane ramnopholane

labdane pimarane

abietane kaurane

OPP

clerodane

Examples of diterpenes in Examples of diterpenes in EuphorbiaEuphorbia species species

jatrophane E. platyphyllos

O

O

H

H O

ent-abietaneE. fischeriana

H

H

O

HOO

ent-atisaneE. characias

H

H

HO

OH

ent-trachylobaneE. wallichii

H

H

O

OH

ent-kauraneE. sieboldiana

OHCH

H

HO

OH

ent-isopimaranesE. quinquecostata

O

HHO

OiBu

HH

HO

OH

OAngO

tiglianeE. grandicornis

AcO

AcO OAc

BzO

AcO

H OAc

O

OHOH

H

H

H

jatropholaneE. lagascae

Discovery of diterpenes in Discovery of diterpenes in EuphorbiaEuphorbia species species

• Aim: Aim: identification of the irritant, proinflammatory compounds

• 1935. Böhm: 1935. Böhm: first isolation of phorbol in crystalline form from croton oil

• 1960. Hecker: 1960. Hecker: structure determination of phorbol

• 1937. Dublyanskaya: 1937. Dublyanskaya: first isolation of a macrocyclic diterpene „euphorbiasteroid” from Euphorbia lathyris

• 1970. Zechmeister: 1970. Zechmeister: Structure determination of „euphorbiasteroid” as the lathyrane diterpene L1

phorbol „euphorbiasteroid”

Diterpenes in Euphorbiaceae Diterpenes in Euphorbiaceae species Ispecies I

cembrane casbane jatrophane modified jatrophane

tigliane daphnane ingenane

MonocyclicMonocyclic BicyclicBicyclic

Skeletal types of well known phlogistic compoundsSkeletal types of well known phlogistic compounds„Phorboids”„Phorboids”

TetracyclicTetracyclicTricyclicTricyclic

lathyrane jatrophatrion type jatropholane euforactin A type paraliane

eufactin type crotopholane pepluane euforactin B type segetane

ramnopholane myrsinane euphoreppinol type cyclomyrsinane

Diterpenes in Diterpenes in EuphorbiaceaeEuphorbiaceae species IIspecies II

Why are Why are EuphorbiaEuphorbia diterpenes interesting?diterpenes interesting?

• High biological activity of the compounds• Many uninvestigated plants • Structural diversity, promising source for

finding new chemical entities• Interesting targets of drug discovery• Chemotaxonomic significance (markers)

prostratinreactivate latent HIV virus

phase I clinical trials

HOHOO CH2OH

O H

H

O

O

O

O

OHO

H

H

O OH

OCH3

O

O

HHO

OAc

HH

HO

OH

HO

BzO

OAc

HOAc

HOAc

OAc

O

Ingenol 3-angelate (PicatoIngenol 3-angelate (Picato®®))treatment of keratosistreatment of keratosis

pepluanonanti-inflammatorypreclinical phase

resiniferatoxinultrapotent TRPV-1 agonistphase II and III clinical trials

Drug discovery and Drug discovery and EuphorbiaEuphorbia diterpenes diterpenes

Screened Euphorbiaceae species Screened Euphorbiaceae species (32)(32)

Hungarian species Hungarian species DC Place of collectionDC Place of collection

Mercurialis perennis – Vértes montain

Euphorbia angulata Szent György montain

E. amygdaloides – Mecsek montain

E. cyparissias + Tápé

E. esula + Szeged

E. exigua + Mosonmagyaróvár

E. falcata + Orosháza

E. helioscopia + Szeged

E. lucida – Makó

E. maculata – Debrecen

E. palustris Kiszombor

E. pannonica + Isaszeg

E. peplus + Pesthidegkút

E. platyphyllos + Csáfordjánosfa

E. polychroma Hármashatárhegy

E. salicifolia + Pesthidegkút

E. segueriana + Ásotthalom

E. segueriana + Budaőrs

ssp. minor

Hungarian species Hungarian species DC Place of collectionDC Place of collection

E. serrulata + Iklódbördöce

E. virgata Budaőrs

E. villosa + Vácrátót

Not native, ornamental plantsNot native, ornamental plants

E. davidii – Szeged

E. dentata – Igal

E. grandicornis + Szeged

E. lathyris + Székesfehérvár

E. myrsinites + Szeged

E. abyssinica Szeged

Outlander plantsOutlander plants

Acalypha fruticosa + Jemen

E. chamaesyce – Croatia

E. hirta – Zanzibar

E. mongolica + Mongolia

E. terracina + Crete

DC = Diterpene content investigated by TLC

Screening for diterpene Screening for diterpene content of Euphorbiaceae content of Euphorbiaceae

speciesspecies

1. Extraction with MeOH (10x)2. Concentration (1/10)3. Extraction with CH2Cl2

Plant material

Dichloromethane fraction

Aqueous MeOH fraction

MeOH – H2O (3:2, 4:1, 1:0

v/v)TLC investigation

Sorbent: Kieselgel 60F254

Developing system: A. cyclohexane – EtOAc – EtOH (60:30:1)B. CHCl3 – acetone (19:1)Detection: ccH2SO4 + 10’ 105 °C

A B

1. E. platyphyllos CH2Cl2 extract2. Polyamide 60% MeOH fraction3. Polyamide 80% MeOH fraction 4. Polyamide MeOH fraction

Polyamide

10 Plant species selected for 10 Plant species selected for preparative workpreparative work

Euphorbia esula undried whole plant

Euphorbia peplus undried whole plant

Euphorbia lathyris undried roots

Euphorbia serrulata undried whole plant

Euphorbia salicifolia undried whole plant

Euphorbia platyphyllos dried whole plant

Euphorbia mongolica dried whole plant

Euphorbia falcata undried whole plant

Euphorbia exigua undried whole plant

Euphorbia pannonica undried whole plant

Isolation strategy of Isolation strategy of Euphorbia Euphorbia diterpenesditerpenes

ExtractionExtraction1. Fresh plant material: Percolation with MeOH 2. Solvent-solvent partition

Apolar extract

Crude separationsCrude separations

1. Open column chromatography on polyamide (ICN) mobile phase: MeOH-H2O (2:3, 3:2, 4:1, 1:0)

Diterpene fraction

2. Vacuum liquid chromatography: sorbent Kieselgel 60, eluent: a/ hexane-EtOAc-EtOH b/ petrol-EtOAc c/ cyclohexane-acetone d/ CHCl3-acetone e/ cyclohexane-EtOAc gradient systemsk

1. Dried plant material: Percolation with CHCl3 2. Concentration

Isolation of diterpenes from Isolation of diterpenes from Euphorbia Euphorbia species IIspecies II

1. Open column chromatography on RP-silica Sorbent: LiChroprep RP-18 2. Vacuum liquid chromatography Sorbent: Kieselgel 60 3. Centrifugal partition chromatography System: nHexane-EtOAc-MeCN-MeOH (8:2:2:3)4. Preparative and centrifugal TLC Sorbent: Kieselgel 60 5. NP-HPLC, RP-HPLC Column: LiChrospher Si 100, LiChrospher RP-18

Solvent systems On NP-silica: On RP-silica: benzene-EtOAc MeOH-H2O

chloroform-acetone MeCN-H2O chloroform-MeOH cyclohexane-EtOAc-EtOHnhexane-tetrahydrofuran-acetonedichloromethane-acetonenhexane-EtOAc

benzene-chloroform-diethylether

AcO

AcO

R1O

AcOHAcO

OAc

O

CH2

OR2

O

HH

R1 R2

A iBu AcB Ac iBuC iBu iBu

LiChrospher Si 100; cyclohexane-EtOAc-EtOH 20:10:1; 0.3 ml/min; RI detection

Fine separationsFine separations

Diterpenes isolated from E. serrulata

Structure elucidation IStructure elucidation I

HRESIMS:HRESIMS: PL-2 m/z 798,1902 (M+Cs)+, C35H44O12

1H NMR: (CDCl3, 500 MHz)

1x benzoyl

4x acetyl 5x methyl

skeletal protons

JMOD spectrum: JMOD spectrum: (CDCl3, 125 MHz) ester groups + 20 carbon atoms

Quaternary C/CH2

204.0137.990.673.741.6

40.1

CH/CH3

141.6141.6127.277.573.775.867.643.240.027.925.818.018.517.0

Structure elucidation IIStructure elucidation II

HMQC spectrum: HMQC spectrum: assignment of protons and protonated carbons

1H NMR 13C NMR ( ppm)

1x -CH2-40.1 2.71 dd, 2.05 d9x -CH-127.2 5.73 dd 141.,6 5.99 brs141.6 5.47 d 77.5 5.86 brd 75.8 5.27 brs73.7 5.67 s67.6 5.39 brs43.2 3.70 dq 40.0 2.33 m5x -CH3

25.8 0.91 s27.9 1.13 s18.5 1.25 d 18.0 0.87 s17.0 1.43 d Quaternary carbons204.0 -137.9 -90.6 -73.7 -41.6 -OH 3.01 s

Structure elucidation IIIStructure elucidation III

11H-H-11H COSY spectrum: H COSY spectrum: identification of partial structures

CHCH

OR

3x -CH3

CHCH

OROR

CH

CH3

CH CH

CHCH2

CH3

CH

OR

Structure elucidation IVStructure elucidation IV

HMBC spectrum:HMBC spectrum: connection of structural fragment

HMBC correlations CH1H,1H COSY correlations

C-10

C-15

C-4

C-6

C-14

O

AcO

O

O

OO

HO

O

OO

O

CH2

CH

CH

CH3

CHCH3

CHCH

CHCH

CH

-CH3

-CH3

-CH3

Structure elucidation VStructure elucidation V

NOESY spectrum: NOESY spectrum: determination of stereochemisty8 stereogenic center in Pl-2!

HO

AcOOAc

O

BzO

AcO

OAc

12

43 5

6

7 8

9

1112

1315

16

17

18

19

NOEs indicating α positionsH-3 H-17H-3 H-7H-3 H-8H-3 H-1b()H-1b() H-13H-1b() H-16

NOEs indicating β positions15-OAc H-2’,6’6-OH H-9

E geometry of C-4/C-5 H-5 15-OAcH-5 H-11

Diagnostic Overhauser effects

Structure elucidation VIStructure elucidation VI

Structure elucidation VII. X-ray Structure elucidation VII. X-ray diffraction – absolute diffraction – absolute

configurationconfiguration

Pepluane diterpeneEuphorbia peplus

Conformer I Conformer II

EuphorbiaEuphorbia esulaesula

AcO

AcOAcOH

O

CH2

AcO

OAc

NicO

ONic

OAcO

AcO

AcO

AcOH

OAc

OH

O

H

H

OiBu O

O

OAcO

AcO

AcO

AcOH O

CH2

OAcO

AcO

AcO

AcOH

OAc

OAc

CH2

O

H

H

OiBu

OAcO

AcO

R2

R1

AcOH

CH2

R4

R3

OAcO

AcO

R2

R1

AcOH

CH2

O

O

CH2

O

AcOH

AcO

AcO

HAcO OAc

OAc

HO

OAc

iBu

R1 R2 2 OBz OAc 3 H iBu

10 OAc OAc

R1 R2 R3 R4 4 ONic OAc H ONic 5 OAc OiBu H ONic 6 H OiBu H ONic 7 OAc OAc H ONic

11 OAc OAc H OAc 12 OAc OiBu OAc OAc

14

13

9

81

Ac=acetyl, iBu=isobutanoyl, Bz=benzoyl, Nic=nicotinoyl

Hohmann, J., Vasas, A., Günther, G., Máthé, I., Evanics, F., Dombi, Gy., Jerkovich, Gy. J. Nat. Prod. 60, 331-335 (1997); Günther, G., Hohmann, J., Vasas, A., Máthé, I., Dombi, Gy., Jerkovich, Gy. Phytochemistry 47, 1309-1313 (1998); Günther, G., Martinek, T., Dombi, Gy., Hohmann, J., Vasas, A. Magn. Reson. Chem. 37, 365-370 (1999); Vasas, A., Sulyok, E., Rédei, D., Forgo, P., Szabó, P., Zupkó, I., Berényi, Á., Molnár, J., Hohmann, J. J. Nat. Prod. 74, 1453-1461 (2011)

EuphorbiaEuphorbia salicifoliasalicifolia

AcO

R1O

AcO

HAcO

OAc

AcO

OR2

O

H

H

O

AcO

AcO

AcO

HAcO

OAc

iBuO

OAc

O H

O

HO

R1 R2

17 iBu iBu18 Ac iBu13 iBu Ac

AcO

AcOAcOH

O

CH2

AcO

OAc

NicO

ONic

9

OAcO

AcO

AcO

AcOH

OAc

OH

O

H

H

OiBu O

O

8

19

Hohmann, J., Evanics, F., Dombi, Gy., Szabó, P. Tetrahedron Lett. 42, 6581-6584 (2001); Hohmann, J., Evanics, F., Dombi, Gy., Molnár, J., Szabó, P. Tetrahedron 57, 211-215 (2001)

EuphorbiaEuphorbia pepluspeplus

BzO

OH

OAc

OR2

HOAc

OR1

OAc H

BzO

R1O

HO

HAcO

OR3

AcO

OR2

AcO

R1 R2 R1 R2 R3

20 Ac Ac 23 iBu H Nic21 Ac H 24 Ac Ac Ac22 H Ac 25 iBu H Ac

26 Ac H NicO

R6O

R2O

R4O

R1

R3OH OR5

OAc

O

R3

H

H

H

HOR2O

R1O

R1 R2 R3 R4 R5 R6 R1 R2 R3

27 OAc Bz Ac iBu Nic H 30 Ang H OH28 H Bz Ac Ac Ac Ac 31 Ang H H29 H Ac Bz Ac Ac Ac 32 H Ang H

Ang=angeloyl

Hohmann, J., Evanics, F., Berta, L., Bartók, T. Planta Med., 66, 291-294 (2000); Hohmann, J., Günther, G., Vasas, A., Kálmán, A., Argay, Gy. J. Nat. Prod. 62, 107-109 (1999); Hohmann, J., Vasas, A., Günther, G., Dombi, Gy., Blazsó, G., Falkay, Gy., Máthé, I., Jerkovich, Gy. Phytochemistry 51, 673-677 (1999)

Diterpenes isolated from Diterpenes isolated from E. peplus E. peplus of different originof different origin

Ingenanes

Jatrophanes

Pepluanes

Germany Egypt Egypt Chile Germany Italy Hungary

EuphorbiaEuphorbia serrulataserrulata

BzO

TigOOAc

OH

OAcAcO

O BzO

ROOAc

OH

OAcAcO

OBzO

AcOOAc

OAc

OAc

AcOBzO

ROOAc

OAc

OAcAcO

HOBzO

AcOOAc

OAc

OAcO

RO

BzO

AcOOAc

OAc

OAcO

AcO

BzO

AcOOAc

OAc

OAcO

AcO

RO

BzO

AcOOAc

OAc

OHO

HO

AcO

BzO

ROOAc

OAc

AcOOAc

H

33 34 R = Tig 36 37 R=Ac 39 R=H 38 R=Bz 40 R=Ac

41 42 R = H 44 45 R=H 43 R = Ac Tig=tiglyl

Hohmann, J., Rédei, D., Evanics, F., Kálmán, A., Argay, Gy., Bartók, T. Tetrahedron 56, 3619-3623 (2000); Hohmann, J., Molnár, J., Rédei, D., Evanics, F., Forgo, P., Kálmán, A., Argay, Gy., Szabó, P. J. Med. Chem. 45, 2425-2431 (2002); Rédei, D., Hohmann, J., Evanics, F., Forgo, P., Szabó, P., Máthé, I. Helv. Chim. Acta 86, 280-289 (2003)

E. pannonicaE. pannonica

O

O

HO

HHHO

H

OiBu

OAng

OH

O

OH

HO

HHHO

H

OiBu

OAng

48

49

E. platyphyllosE. platyphyllos

35 R = Bz

BzO

ROOAc

OH

OAcAcO

O

46 R=Ac

BzO

ROOAc

OAc

AcOOAc

HBzO

AcOOAc

OAc

OAcO

HO

47

39 R = H

BzO

AcOOAc

OAc

OAcO

RO

Hohmann, J., Forgo, P., Csupor, D., Schlosser, G. Helv. Chim. Acta 86, 3386-3393 (2003); Sulyok, E., Vasas, A., Rédei, D., Dombi, G., Hohmann, J. Tetrahedron 65, 4013-4016 (2009)

Euphorbia Euphorbia villosavillosa

OHHAcO

BzO

RO

H

H

O

HOH

H

H

AcO

BzOO

OMe

O

HO

H

HBzO

AcO

O

H

H

HAcO

AcOAcO

50 R = Me 5251 R = H

53 54 55

BuO

HO

HAcO

HOO

H

H

E. lathyrisE. lathyris

Vasas, A., Hohmann, J., Forgo, P., Szabó, P. Tetrahedron 60, 5025-5030 (2004); Hohmann, J., Evanics, F., Vasas, A., Dombi, Gy., Jerkovich, Gy., Máthé, I. J. Nat. Prod. 62, 176-178 (1999)

EuphorbiaEuphorbia falcatafalcataAcO

AcOR2O

AcO

OH

H

H

H

OAcR1

O OR3

R1O

R4OAcO

H

HR2O

OH

H

R3

H

AcOAcO

HAcO

H

H

O

OAcH OBzRO

R62 Prop63 iBu

R1 R2 R3

56 OBz iBu MeBu57 OBz iBu iBu58 H Prop iBu

R1 R2 R3 R4

59 Hex H H H60 Prop Ac OBz Ac61 iBu H OBz Ac

AcO

BzOAcO

HO

OH

H

H

H

OAc

O OBz

R1 R2 R3 R4

64 Ac Bz Bz H65 iBu Ac Ac Ac

R66 Prop67 iBu

AcO

AcO

AcO

OHH

H

O

OAc

RO

Prop=propanoylHex=hexanoylMeBu=2-methyl-butanoyl

Vasas, A., Sulyok, E., Martins, A., Rédei, D., Forgo, P., Kele, Z., Zupkó, I., Molnár, J., Pinke, G., Hohmann, J. Tetrahedron 68, 1280-1285 (2012); Sulyok, E., Vasas, A., Rédei, D., Forgo, P., Kele, Z., Pinke, G., Hohmann, J. Tetrahedron 67, 7289-7293 (2011)

Euphorbia grandicornisEuphorbia grandicornis

Forgo, P., Rédei, D., Hajdu, Zs. Szabó, P., Szabó, L., Hohmann, J. J. Nat. Prod. 74, 639-643 (2011)

O

HHO

OiBu

HH

HO

OAng

1

2

3 4

5 6

7

89

10

11

12

13

14

15

16

17

18

19

20

O

HHO

OiBu

HH

HO

OR

OAc

O

HHO

OR1

HH

HO

OR2

O

HHO

OiBu

HH

HO

OH

OAngO

68 R=H69 R=Ang

70 R1=Ac, R2=H71 R1=iBu, R2=H72 R1=iBu, R2=Ac73 R1=MeBu, R2=Ac

74 75

Chemotaxonomic significance of Chemotaxonomic significance of EuphorbiaEuphorbia diterpenes - diterpenes -

morphologymorphology

E. platyphyllos E. serrulata capsule covered with hemispherical - cylindrical tubercles

E. platyphyllosE. platyphyllos

Pl-1 Pl-3

Eser-1 R1=CH3 R2=HEser-2 R1=H R2=CH3

Eser-4 Eser-9

Pl-4

Eser-8= Pl-2

E. serrulataE. serrulata

OH

AcO

BzO O

OAc

OAcBzO

HO

AcOOAc

O

BzO

AcO

OAc

AcO

BzO

AcO

H

OAc

AcO

OAc

HO

AcOOAc

OAc

BzO

AcO

OAc

OH

AcO

R1

BzO O

OAc

OAcTigO

R2

AcO

BzO

HO

H

OAc

AcO

OAc

OAc

AcO

BzO

OAc

OAcAcO

HO

Chemotaxonomic significance of Chemotaxonomic significance of EuphorbiaEuphorbia diterpenes - diterpenes -

chemistrychemistry

Multidrug resistance reversal Multidrug resistance reversal activity of diterpenesactivity of diterpenes

• Multidrug resistance reversing activity:– L5178 Mouse lymphoma cells transfected by pHa MDR1/A

retrovirus

– Rhodamine 123 exclusion test

– Evaluation by flow cytometry using Becton Dickinson FACScan instrument

– Fluorescence activity ratio (R) was calculated

1 2

OAcO

AcO

AcO

AcOH

CH2

ONic

iBuO

OAcO

AcOAcOH

CH2

ONic

iBuO3

OAcO

AcOAcOH

CH2

O

iBuO

Molnár, J., Engi, H., Hohmann, J., Molnár, P., Deli, J., Weselowska, O., Michalak, K., Wang, Q. Curr. Top. Med. Chem. 10, 1757-1768 (2010); Vasas,A.,Rédei, D., Csupor, D., Molnár, J., Hohmann, J. Eur. J. Org. Chem. 5115-5130 (2012)

g/mlg/ml

R

■ 10 µg/ml

• Euphorbia peplus metabolite ingenol-mebutate = ingenol 3-angelate

• FDA appoval: 26 January 2012• Indication: actinic (solar) keratosis• Planed European introduction• Picato® gel 0.015% and 0.05%

Picato® (PEP005) - a new plant chemotype in the therapy

First experiment in Szeged

1997. Collection of plant material in Pesthidegkút, 900 g fresh herb

1997-2000. Isolation of diterpenes (13 compounds) from E. peplus

7 jatrophanes 3 pepluanes Ingenanes: PEP005, Pe-2, Pe-3

Publications Hohmann J., Evanics F., Berta L., Bartók T. Planta

Med. 2000, 66, 291-293 Hohmann J, Günther G, Vasas A, Kálmán A, Argay

G. J. Nat. Prod. 62, 107 (1999) Hohmann J, Vasas A, Günther G, Dombi G, Blazsó

G, Falkay G, Máthé I, Jerkovich G. Phytochemistry 51, 673 (1999)

Pharmacology of ingenane diterpenes

Pharmacological investigations: Proinflammatory activity of the extracts and compounds

on mouse ear test E. peplus extract: IC50 25 µg/ear Jatrophane diterpenes: inactive

Literature data: Proinflammatory activity on mouse ear test:

PEP005: IC50 0.04 nmol/ear (4h), 0.12 nmol/ear (24h) Pe-2: IC50 0.17 nmol/ear, 0.48 nmol/ear (24h) Pe-3: IC50 10 nmol/ear, >100 nmol/ear (24h)

Co-carcinogenic effect on in vivo mouse skin model: PEP005: tumor yield 0/26 (12 weeks), 0/10 (24 weeks) Pe-1: tumor yield 6/25 (12 weeks) Pe-2: tumor yield 0/28 (12 weeks), 2/27 (24 weeks)

Hohmann J, Vasas A, Günther G, Dombi G, Blazsó G, Falkay G, Máthé I, Jerkovich G. Phytochemistry 51, 673 (1999); Gotta H, Adolf W, Opferkuch HJ, Hecker E Z Naturfortsch 39b, 683 (1984); Salah MAD, Farghaly ZM, Taha H, Gotta H, Hecker E J Cancer Res Clin Oncol 124, 131 (1998)

HOHOO CH2OH

O H

H

O

PEP005

HOHOO

O H

H

O

Pe-2

OHO

HO

O H

H

O

Pe3

New informations…New informations… Ingenol esters act as protein kinase C (PKC) activators

PKC isoenzyme selectivity in vivo α-isoform: antiapoptotic -isoform: antiproliferative, proapoptotic activity

PEP005: selective effects on -isoforms, induction of nuclear translocation of PKC isoenzyme

2004. Preclinical development 2005. Clinical trials >1000 patients Euphorbia diterpenes in 44 patents!

Treatment of solid cancers, melanomas, squammous carcinomas and prostate cancer

Treatment and prophylaxis of acne vulgaris

Kedei N, Lundberg DJ, Tóth A, Welburn P, Garfield SH, Blumberg PM. Cancer Res 64, 3243 (2004)

• Isolation from plants: Isolation from plants: – E. peplus, E. antiquorum, E. paralias,

E. helioscopia, E. drummondii, E. hirta• Production from Production from E. peplusE. peplus

– Cultivation, harvesting– Extraction, fractionation, purification– Formulation

• By semisynthesisBy semisynthesis– From E. lathyris ingenol– Esterification

• Total synthesis (4 methods)Total synthesis (4 methods)

Production of PEP005Production of PEP005

ingenol-3-angelát PEP005

HOHOO CH2OH

O H

H

O

SummarySummary Middle-European flora is still promising source of drug

discovery Known compound may also be interesting Pharmacological investigations turned the judgement

of a molecule Complex structure is not a problem, production may be

made by isolation

Thanks to:Andrea Vasas

Dóra RédeiDezső Csupor

Thank you for your kind attention!