Judit Hohmann
Institute of Pharmacognosy, University of Szeged
DITERPENES FROM EUROPEAN EUPHORBIA
SPECIES SERVING AS PROTOTYPES FOR
NATURAL-PRODUCT-BASED DRUG DISCOVERY
University of Szeged, Faculty of
Pharmacy
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
University of Szeged
Faculty of Music
Faculty of Science and Informatics
Juhász Gyula Teacher Training Faculty
Faculty 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 Pharmacognosy
• 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 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 family
• Euphorbiaceae family:
– around 7.500 species in 300 genera
– morphology: non-succulent, succulent or cactiform
– herbs, shrubs or trees
• Widely used Euphorbiaceae 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
Euphorbia genera
• Number 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
– 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 diterpenes
„Lower terpenes”
Macrocyclic
diterpenes and their
cyclisation products
OPP
OPP
„Higher terpenes”
Cyclic diterpenes
GGPP
cembrene cation
lathyrane
labdane
jatrophane
clerodane
ingenane
daphnane
pimarane
tigliane
ramnopholane
abietane
kaurane
Examples of diterpenes in Euphorbia
species
jatrophane
E. platyphyllos
tigliane
E. grandicornis
jatropholane
E. lagascae
ent-abietane
E. fischeriana
ent-atisane
E. characias
ent-trachylobane
E. wallichii
ent-kaurane
E. sieboldiana
ent-isopimaranes
E. quinquecostata
Discovery of diterpenes in Euphorbia
species
• Aim: identification of the irritant, proinflammatory
compounds
• 1935. Böhm: first isolation of phorbol in crystalline
form from croton oil
• 1960. Hecker: structure determination of phorbol
• 1937. Dublyanskaya: first isolation of a macrocyclic
diterpene „euphorbiasteroid” from Euphorbia lathyris
• 1970. Zechmeister: Structure determination of
„euphorbiasteroid” as the lathyrane diterpene L1
phorbol
„euphorbiasteroid”
Diterpenes in Euphorbiaceae species I
Skeletal types of well known phlogistic compounds
„Phorboids”
tigliane
daphnane
Monocyclic
cembrane
ingenane
Bicyclic
casbane
jatrophane
modified jatrophane
Diterpenes in Euphorbiaceae species II
Tricyclic
lathyrane
eufactin type
ramnopholane
jatrophatrion type
crotopholane
myrsinane
Tetracyclic
jatropholane
pepluane
euforactin A type
euforactin B type
euphoreppinol type
paraliane
segetane
cyclomyrsinane
Why are Euphorbia 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)
Drug discovery and Euphorbia
diterpenes
O
OAc
HO
H
O
O
H
H
H
H
O HO
O
OH
OCH3
O
OH
O
prostratin
reactivate latent HIV virus
phase I clinical trials
OH
resiniferatoxin
ultrapotent TRPV-1 agonist
phase II and III clinical trials
H
OAc
HO
OAc
H
O
OAc
B zO
H
O
H
OAc
O
O
C H 2O H
HO
HO
pepluanon
anti-inflammatory
preclinical phase
Ingenol 3-angelate (Picato®)
treatment of keratosis
Screened Euphorbiaceae species (32)
Hungarian species
Mercurialis perennis
Euphorbia angulata
E. amygdaloides
E. cyparissias
E. esula
E. exigua
E. falcata
E. helioscopia
E. lucida
E. maculata
E. palustris
E. pannonica
E. peplus
E. platyphyllos
E. polychroma
E. salicifolia
E. segueriana
E. segueriana
ssp. minor
DC
–

–
+
+
+
+
+
–
–

+
+
+

+
+
+
Place of collection
Vértes montain
Szent György montain
Mecsek montain
Tápé
Szeged
Mosonmagyaróvár
Orosháza
Szeged
Makó
Debrecen
Kiszombor
Isaszeg
Pesthidegkút
Csáfordjánosfa
Hármashatárhegy
Pesthidegkút
Ásotthalom
Budaőrs
Hungarian species
E. serrulata
E. virgata
E. villosa
DC
+

+
Place of collection
Iklódbördöce
Budaőrs
Vácrátót
Not native, ornamental plants
E. davidii
–
Szeged
E. dentata
– Igal
E. grandicornis
+ Szeged
E. lathyris
+ Székesfehérvár
E. myrsinites
+ Szeged
E. abyssinica
 Szeged
Outlander plants
Acalypha fruticosa
E. chamaesyce
E. hirta
E. mongolica
E. terracina
DC = Diterpene content investigated by TLC
+
–
–
+
+
Jemen
Croatia
Zanzibar
Mongolia
Crete
Screening for diterpene content of
Euphorbiaceae species
Plant material
1. Extraction with MeOH (10x)
2. Concentration (1/10)
3. Extraction with CH2Cl2
Dichloromethane
fraction
Polyamide
MeOH – H2O
(3:2, 4:1, 1:0
v/v)
Aqueous MeOH
fraction
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 extract
2. Polyamide 60% MeOH fraction
3. Polyamide 80% MeOH fraction
4. Polyamide MeOH fraction
10 Plant species selected for
preparative work
Euphorbia esula
Euphorbia peplus
Euphorbia lathyris
Euphorbia serrulata
Euphorbia salicifolia
Euphorbia platyphyllos
Euphorbia mongolica
Euphorbia falcata
Euphorbia exigua
Euphorbia pannonica
undried whole plant
undried whole plant
undried roots
undried whole plant
undried whole plant
dried whole plant
dried whole plant
undried whole plant
undried whole plant
undried whole plant
Isolation strategy of Euphorbia
diterpenes
Extraction
1. Fresh plant material:
Percolation with MeOH
2. Solvent-solvent partition
1. Dried plant material:
Percolation with CHCl3
2. Concentration
Apolar extract
Crude 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
Isolation of diterpenes from Euphorbia
species II
Fine separations
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
O
AcO
O
H
H
C H2
AcO
AcO
H
AcO
O Ac
R1 O
A
B
C
R1
iBu
Ac
iBu
O R2
R2
Ac
iBu
iBu
LiChrospher Si 100; cyclohexane-EtOAcEtOH 20:10:1; 0.3 ml/min; RI detection
Solvent systems
On NP-silica:
benzene-EtOAc
chloroform-acetone
chloroform-MeOH
cyclohexane-EtOAc-EtOH
nhexane-tetrahydrofuran-acetone
dichloromethane-acetone
nhexane-EtOAc
benzene-chloroform-diethylether
On RP-silica:
MeOH-H2O
MeCN-H2O
Diterpenes isolated from E. serrulata
Structure elucidation I
HRESIMS: PL-2 m/z 798,1902 (M+Cs)+, C35H44O12
1H
NMR: (CDCl3, 500 MHz)
1x benzoyl
skeletal protons
4x acetyl
5x methyl
Structure elucidation II
JMOD spectrum: (CDCl3, 125 MHz) ester groups + 20 carbon atoms
Quaternary C/CH2
204.0
137.9
90.6
73.7
41.6
40.1
CH/CH3
141.6
141.6
127.2
77.5
73.7
75.8
67.6
43.2
40.0
27.9
25.8
18.0
18.5
17.0
Structure elucidation III
HMQC spectrum: assignment of protons and protonated carbons
1H
NMR
13C
NMR ( ppm)
1x -CH240.1
2.71 dd, 2.05 d
9x -CH127.2
141.,6
141.6
77.5
75.8
73.7
67.6
43.2
40.0
5.73 dd
5.99 brs
5.47 d
5.86 brd
5.27 brs
5.67 s
5.39 brs
3.70 dq
2.33 m
5x -CH3
25.8
0.91 s
27.9
1.13 s
18.5
1.25 d
18.0
0.87 s
17.0
1.43 d
Quaternary carbons
204.0
137.9
90.6
73.7
41.6
OH
3.01 s
Structure elucidation IV
1H-1H
COSY spectrum: identification of partial structures
CH2
CH
CH
CH3 OR
CH
CH
CH
CH3
CH
CH
OR
OR
CH
CH
OR
3x -CH
3
Structure elucidation V
HMBC spectrum: connection of structural fragment
AcO
O
CH
CH
CH2
CH3 CH
CH
O
CH3
CH
-CH3
C-10
-CH3
O
O
HO
-CH3
O
CH
O
O
O
O
C-6
C-15
CH
CH
C-4
HMBC correlations CH
1H,1H COSY correlations
C-14
Structure elucidation VI
NOESY spectrum: determination of stereochemisty
8 stereogenic center in Pl-2!
Diagnostic Overhauser effects
NOEs indicating α positions
H-3
H-17
H-3
H-7
H-3
H-8
H-3
H-1b()
H-1b()
H-13
H-1b()
H-16
NOEs indicating β positions
15-OAc
H-2’,6’
6-OH
H-9
16
E geometry of C-4/C-5
H-5
15-OAc
H-5
H-11
O
Ac O
13
1
2
15
12
4
3
B zO
17
11
5
6
9
HO
7
Ac O
18
8
O Ac
19
O Ac
Structure elucidation VII. X-ray
diffraction – absolute configuration
Pepluane diterpene
Euphorbia peplus
Conformer I
Conformer II
Euphorbia esula
R
1
R
2
1
O Bz
R
1
R
2
R
3
R
4
4
O N ic
OAc
H
O N ic
5
OAc
O iB u
H
O N ic
6
H
O iB u
H
O N ic
7
OAc
OAc
H
O N ic
11
OAc
OAc
H
OAc
12
OAc
O iB u
OAc
OAc
2
8
13
OAc
3
H
iB u
10
OAc
OAc
Ac=acetyl, iBu=isobutanoyl,
Bz=benzoyl, Nic=nicotinoyl
9
14
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)
Euphorbia salicifolia
17
18
13
R1
iBu
Ac
iBu
9
R2
iBu
iBu
Ac
19
8
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)
Euphorbia peplus
R1
Ac
Ac
H
20
21
22
R2
Ac
H
Ac
23
24
25
26
R1
iBu
Ac
iBu
Ac
R2
H
Ac
H
H
R3
Nic
Ac
Ac
Nic
Ang=angeloyl
27
28
29
R1
OAc
H
H
R2
Bz
Bz
Ac
R3
Ac
Ac
Bz
R4
iBu
Ac
Ac
R5
Nic
Ac
Ac
R6
H
Ac
Ac
30
31
32
R1
Ang
Ang
H
R2
H
H
Ang
R3
OH
H
H
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 E. peplus
of different origin
Germany
Ingenanes
Jatrophanes
Pepluanes
Egypt
Egypt
Chile
Germany
Italy
Hungary
Euphorbia serrulata
33
34
41
R = Tig
42
43
R=H
R = Ac
36
37 R=Ac
38 R=Bz
44
39 R=H
40 R=Ac
45 R=H
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. platyphyllos
35 R = Bz
46 R=Ac
39 R = H
47
E. pannonica
48
49
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 villosa
50 R = Me
51 R = H
52
E. lathyris
53
54
55
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)
Euphorbia falcata
56
57
58
R1
OBz
OBz
H
R2
iBu
iBu
Prop
64
65
R1
Ac
iBu
R3
MeBu
iBu
iBu
R2
Bz
Ac
R1
R2
Hex H
Prop Ac
iBu H
59
60
61
R3
Bz
Ac
R4
H
Ac
R3
H
OBz
OBz
R4
H
Ac
Ac
66
67
62
63
R
Prop
iBu
R
Prop
iBu
Prop=propanoyl
Hex=hexanoyl
MeBu=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 grandicornis
OR1
OiBu
HO
H
HO
H
H
H
OR
H
H
O HO
O HO
OR2
OAc
70
71
72
73
68 R=H
69 R=Ang
R1=Ac, R2=H
R1=iBu, R2=H
R1=iBu, R2=Ac
R1=MeBu, R2=Ac
OiBu
18
HO
H
H
H
O
O HO
HO
H
1
OAng
19
2
3
4
O HO
OH
10
OiBu
12
11
9
13
8
14
H
7
5
74
Forgo, P., Rédei, D., Hajdu, Zs. Szabó, P., Szabó, L., Hohmann, J. J. Nat. Prod. 74, 639-643 (2011)
6
75
15
20
H
17
16
OAng
Chemotaxonomic significance of
Euphorbia diterpenes - morphology
E. platyphyllos
capsule covered with hemispherical -
E. serrulata
cylindrical tubercles
Chemotaxonomic significance of
Euphorbia diterpenes - chemistry
Ac O
E. serrulata
O Ac
Ac O
Ac O
R1
O Ac
Ac O
R2
B zO
O
B zO
O
Ac O
HO
HO
B zO
HO
O Ac
O Ac
Ac O
Eser-1 R1=CH3 R2=H
Eser-2 R1=H R2=CH3
B zO
O Ac
OH
Ti g O
Ac O
H
Eser-4
O Ac
O Ac
Eser-9
O Ac
O Ac
Eser-8
= Pl-2
Ac O
O Ac
B zO
Ac O
Ac O
O
E. platyphyllos
B zO
Pl-1
B zO
OH
Ac O
H
O Ac
Ac O
O Ac
O Ac
B zO
HO
Ac O
O Ac
Pl-3
Pl-4
O Ac
O Ac
Multidrug resistance reversal
activity 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
O
O
A cO
CH2
A cO
R
AcO
CH2
A cO
H
AcO
O
A cO
H
A cO
O N ic
iB u O
iB u O
1
2
O
A cO
g/ml
g/ml
■ 10 µg/ml
CH2
AcO
3
H
A cO
O N ic
iB u O
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)
Picato® (PEP005) - a new plant
chemotype in the therapy
• 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%
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)
H
PEP005
O
H
O
O
HO
C H 2O H
HO
H
Pe-2
O
H
O
O
HO
HO
H
O
Pe3
HO
HO
O
O
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)
H
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!
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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)
Production of PEP005
• Isolation from plants:
– E. peplus, E. antiquorum, E. paralias,
E. helioscopia, E. drummondii, E. hirta
• Production from E. peplus
– Cultivation, harvesting
– Extraction, fractionation, purification
– Formulation
• By semisynthesis
– From E. lathyris  ingenol
– Esterification
• Total synthesis (4 methods)
H
O
ingenol-3-angelát
PEP005
H
O
O
HO
HO
C H 2O H
Summary
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
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Thanks to:
Andrea Vasas
Dóra Rédei
Dezső Csupor
Thank you
for your kind
attention!