diterpenes from european euphorbia species serving as prototypes for natural-product-based drug...
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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!