Rasoanaivo Phragmalin limonoids from Neobeguea

2nd International Symposium
on Natural Products
Cape Town, South Africa
September 23rd – 25th 2014

Research activities in our laboratory
Ethnobotany-based approach
1- Malaria and the immune system
2- Brain behaviour
Biodiversity-based approach
Bioprospecting: random collecting,
extraction, random screen

Title of my presentation:
Phragmalin limonoids from Neobeguea mahafalensis :
isolation, semisynthesis, biological activities.

Philippe Rasoanaivo
University of Antananarivo,
Institut Malgache de Recherches Appliquées (IMRA)

Content
 Brief history of the study.
 Isolation and structure elucidation of limonoids
with sexual enhancing effects.
 Semisynthesis
 Serendipity discovery:
- Aggressive effects;
- Sleep-induced activities.
 Chemical variations in evolving ecosystems.

Madagascar has a unique biodiversity

Biggest baobab

Famous baobabs

This unique biodiversity is associated
with rich cultural heritage

This empirical practice called Sikidy
is used to diagnose diseases.
We contacted this traditional healer
for fortifying plants.
He told us to go to this place.

We went there and found, not a fortifying plant,
but a common plant used to treat erectile dysfunction

We collected roots
Neobeguea mahafalensis (Meliaceae)

Stem barks are used to treat back pains.
In fact talking directly about sex is taboo.
Local community uses “back pains”.
Roots are not used for medical purposes.

Taking advantage of opportunities

I met Prof Jarl Wikberg in Bloomfontein in 2004 at the
38th Congress of the South African Pharmacology Society.
We agreed to investigate the plant. It was the starting point of
very fruitful and successful collaboration.

What tests to use to
demonstrate activities?
In vitro or in vivo?
In the next slide, I give a basic background
of the drug discovery approaches.

Reverse pharmacology

UP

Synergy:
Metabolism

Bio-informatics
In silico

Virtual screening

In enzymo Combinatorial chemistry
HTS
In cellulo
In vitro (isolated organs)
Traditional
Medicinal chemistry
In vivo

Polypharmacology

In homo

BOTTOM

TOP

REDUCTIONIST

Today drug discovery

Chronology

Drug discovery approaches

Observational studies
Traditional knowledge

HOLISTIC

DOWN

We decided to use
the mating behaviour tests

Isolation of bioactive constituents
Dried and powdered roots
Hot water

Aqueous extract (active in mating behaviour tests)
Water-chloroform partition

Water fraction
Uppsala University

Chloroform fraction (active)
Repeated chromatography
Bioactive constituents

Novel limonoids withC-16/C-30 -lactone ring
O

O

O
O

O

OH
O

O
O

O

O

OH

O

O

O

O

O

O
O
O

Libiguins

O

O
O

O

O

OH

O

O

PCT Patent WO 2008/145996 A2, December 2008.
Razafimahefa S et al. Planta Med 2014, 80: 306-314.

Delayed and long-lasting effects of libiguin A
(compilation of several tests)

Mounting frequency

Dose-response effects

Lag-phase of
3 day treatment

The effects follow a very distinct pattern

Increasing values

S
sceever
na al
rio
s

Basic requirements in developing
ethnobotany-based drugs

ly

pp
u
s
t

ox
ic

ac

tiv

e

ly

tt

In

pp

No

su

c

h

of

 Toxi



ck

Drug
Discovery



a

S

n
cie
i
f
f
u

La

A

ive
t
c

in

um

ns



in

hu

m

an

s

Time

Solving the problem of supply
Five alternatives
Wild
collecting

Cultivation

Semisynthesis

Synthesis

Endophyt microorganisms

We chose the semisynthesis
approach

Approach to the semisynthesis
O

O

O

O

O

Trans-lactonisation

O

O

O

Acylation
OH
HO

OH

Phragmalin isolated from
seeds of commercial
Chukrasia tabularis

Libiguin derivatives

Grigorjeva L et al. J Org Chem 2014, 79:4148-53.

OMe
NH-Me

[O]
O

HO

O

O
O O

O

OH

OH
HO

OH

O

KOH

TMSOTf

Libiguin derivatives
Structure-activity relationships

Serendipity discovery:
unexpected aggressive effects
O

O

O

O

O
O

O
O

N
R2

OH
R1
O

O

OH

PCT Patent WO 2013/110744 A2.
Novel compounds, their manufacture and uses.

Serendipity discovery:
unexpected sleep-induced effects
Dried roots (7kg)
Methylene chloride

Methylene chloride extract
Silica gel column chromatography

Three consecutive fractions showed
sleep-induced effects

Major constituent with sleep-induced effects
O

AcO
O

O

O

O

O
O

O
O

O

OH

O

O

Dodoguin

OMe

Other Dodoguin derivatives
O

O
O
O

O

O
O

O
O

O

OH

O

O

OMe

Rare phragmalin limonoids with seco-ring D
17-keto from Neobeguea leandriana

17

Leandreanin B
Coombes et al; J Nat Prod 2003, 66, 735

Phragmalin limonoids and brain behaviour

A plant used to treat
erectile dysfunction
Phragmalin

Libiguin
(Sexual enhancing effects)

(Raw material)

O

AcO
O

O

O

O

O
O

O
O

O

OH

O

OMe

Serendipity

O

Dodoguin

Gidraguin

(sleep-induced effects)

(Aggressive effects)

Exploring wild collecting

Absence of libiguins
in other ecosystems

Another example of chemical variations

Myrothamnaceae family
One genus: Myrothamnus
Two species
M. flabellifolia
(Southern Africa)

M. moschatus
(Madagascar)

Well documented;
Well investigated.

Poorly documented;
Not investigated

Myrothamnus: resurrection plant (inselberg)

Day 0

Day 1

Day 2

Ecology, Traditional uses, Chemical composition

A b u n d a n c e

T IC : 1 3 0 9 1 2 -F G -0 4 .D
2 2 0 0 0 0 0

2 4 .4 3

2 0 0 0 0 0 0
1 8 0 0 0 0 0
1 6 0 0 0 0 0

Caryophyllene oxide.

1 4 0 0 0 0 0
1 2 0 0 0 0 0

2 5 .9 4
1 8 .7 8

1 0 0 0 0 0 0
8 0 0 0 0 0

1 9 .5 2

2 1 . 52 72 . 8 20 4 . 2 1
2 4 .1 3
2 5 .8 1

6 0 0 0 0 0

1 9 .3 3
2 0 .2 7
1 9 .8 9

4 0 0 0 0 0

Confusing
Ethnomedical information

2 8 .3 3

2 6 .9 2

1 8 . 1 91 9 . 8 2

2 4 .9 5
2 8 .0 9
2 3 . 4 72 52 .51
22
.676.32. 58 4
26
132. 7
.8 9
.27
2.668
. 47
66. 3
1..8
220022
.5
2
. 25 42
4 .52
20
11
29 1
.5
.2
17 8
9. 8. 27 1
4 72513.2.33
45
01
..3
03
221111
. 20
1232
27
2.6
7 2626.2
0.331
6
66 5
22
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7
779444
42
0.. 5
1 7 . 41 08 . 7 0 22
. 222672.222
74
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3 2 9 .6
6. 42
. .7
8
79.0
41 .17 6
1 2
2
2001.. 9
.922820 2
2. 575.5.3592327
27
4
1 8 .5 5
1 5 .4 9
.36 . 1
270.7.21
.6
726 58 .28339900.3.8
124
1

2 0 0 0 0 0
3 .7 5

6 .2 7
1 6
5 .52 .616. 8

9 .0 6

5 .0 0

1 0 .0 0

1 5 .0 0

2 0 .0 0

2 5 .0 0

3 3 .3 7

3 0 .0 0

3 5 .0 0

T im e - - >

A b u n d a n c e
T I C : 1 3 0 9 1 2 -F G -0 5 . D
2 2 .8 4

2 2 0 0 0 0 0

-cadinene,
Caryophyllene oxide.

2 0 0 0 0 0 0
1 8 0 0 0 0 0

2 4 .4 1

1 6 0 0 0 0 0
1 4 0 0 0 0 0
1 2 0 0 0 0 0

2 5 .9 4
2 1 .9 0

1 0 0 0 0 0 0

2 2 .5 2
2 52. 6
7 .44 2

8 0 0 0 0 0
2 1 .7 4

6 0 0 0 0 0

1 8 .1 9
2 4 .9 6
2 5 .8 2
2 1 .2 7
2 6 .1 2
2 3 .0 2
1 7 .4 0
2 4 .5 5
1 8 .7 8
2 6 .9 1
2 8 .0 9
51
1 .5 7
1 8 . 7 02 0 . 2
2 5 .21 69 . 2 8
2 0 . 7282 . 1 1
2 8 .3 2
1 9 .5 1
2 3 .3 1
2 6 . 5 42 8 . 2 1
3 .9 4
2
1 .3 7 2 2
1 2
9 0
. 8. 2
3 7
33
2 ..445.8
12 525 . .032
46 . 7 6
2 9 .6 7
2
5
2
2222.22
4
.4 0
9..30
.0
4. .4
2 5 .5
622
2
7
..71
8
77
.2.0
0
22
7
2
8
27
7
629
2
7
.2.3
7
.1
7
74
.2
82
91
.2
95
1 9 . 3 32 1 . 0
2
8
.92
97
2 7
2 . 138. 1 3
3991
0 33
.7 6
2
.090
229

4 0 0 0 0 0
2 0 0 0 0 0

3 .7 5
6 .2 7
5 .0 0

2 4 .2 1

1 0 .0 0

1 5 .0 0

2 0 .0 0

2 5 .0 0

3 0 .0 0

3 5 .0 0

T im e - - >

A b u n d a n c e
T I C : 1 3 0 9 1 2 -F G -0 3 . D

2 4 .4 2

2 0 0 0 0 0 0
1 8 0 0 0 0 0
1 6 0 0 0 0 0

Caryophyllene oxide.

1 4 0 0 0 0 0
2 5 .9 3
1 2 0 0 0 0 0
1 0 0 0 0 0 0
8 0 0 0 0 0

Ignored by local communities

1 9 .3 3
2 4 .2 1

6 0 0 0 0 0

2 1 .5 7
1 9 .8 9

4 0 0 0 0 0

3 .8 0

9 .0 4

5 .0 0

2 6 .4 2
2 5 .8 1
2 6 .9 2

2 4 .9 2
5 6 .4 8
2 5 .2
1 6
9
2 2 .8 0
2 6. .2595
.2
4.46
.81
2 4
2 0 . 2 27 1 . 7 722
232
33
74
4.05
2. 2
3 2
.03
0. .5
6. 9
3
47 2 52 .66 .31 29 72 .86 . 30 9
22 11 ..2
34 2
75
1 8 .1 9
2494
3. .1322724 5. 6. 08
26
0 .2721282.26..2
82 2
1 5
1 ..02250205.3
6 1
2218
18
2 6222
.6
127.7.277.8.02
1 .64. 9
0 0 . 4110881..7
26
1
26.9.2
83
3939
1
1
98 .525 2
220
.37.9
8
43
0520
.171 6. 6 5
.9
.06
2522.2
99
.9
4
5.8
222
7.2748
.6.7
6
.0
1 17 7
.0 1
01
.1512
2.122.68 9

2 0 0 0 0 0

1 0 .0 0

1 5 .0 0

2 0 .0 0

2 5 .0 0

3 0 .0 0

3 5 .0 0

T im e - - >

A b u n d a n c e
T I C : 1 3 0 9 1 2 -F G -0 1 . D
2 1 .6 2

2 6 0 0 0 0 0
2 4 0 0 0 0 0

b-selinene,
Limonene,
trans-p-mentha-1(7),8-dien-2-ol,

2 2 0 0 0 0 0
6 .2 7

2 0 0 0 0 0 0
1 8 0 0 0 0 0
1 6 0 0 0 0 0

1 1 .9 5
1 4 0 0 0 0 0

1 3 .3 9

1 2 0 0 0 0 0
1 0 0 0 0 0 0
9 .4 7
1 0 .0 1

8 0 0 0 0 0
6 0 0 0 0 0

1 2 . 31 63 . 8 7
1 3 .0 5
6 .1 2

4 0 0 0 0 0
2 0 0 0 0 0

3 .7 5
5 .0 0

8 .3 7

1 0 .0 0

2 0 .2 7

1 3 .4 8

1 0 .9 0
1 14 5. 8. 38 3
111
. 22 1. 2 5
01
.1
8 9. 6. 10811
01...56
7 4631 2 . 8 3
3 00
1 6 .4 8
1 5 .0 0

1 1
9 9
. 3. 8
3 8
2 0 .0 0

2 4 .3 6
2 5 2. 86 9. 7 2
7 7. 1 5 2 9 . 3 6
15
872..62
52
.6
84
2
6
4529
..9
2
.0
9 0
2 2 2. 732 5.4622. 41
2 5 .0 0

3 0 .0 0

3 8 .7 9
4 0 .9 3
3 5 .0 0

Convulsions

cis-p-mentha-1(7),8-dien-2-ol.

4 0 .0 0

T im e - - >

trans-pinocarveol, Pinocarvone,
-selinene,
trans-p-mentha-1(7),8-dien-2-ol.

Asthma, cough
Convulsions

Chemical variations in evolving ecosystems:

French botanist collected Gonioma malagasy in 1973
for phytochemical studies

Transition
forest

Wrong structure

Right structure

We isolated 14’,15’-dihydro-pycnanthine
(= pleiomutinine) as the major constituent.
The structure was revised.
P. Rasoanaivo & G. Lukacs; J Org Chem 1976, 41, 376.

Many decades later, we collected the same species in the same
area which was seriously destroyed by deforestation.
We did not find pleiomutinine but another
dimer indole alkaloid we named
Goniomedines A and B (artifact).

2010

Goniomedine A
Gonioma malagasy

Goniomedine B

Beniddir et al. Organic Letters 2012, 14, 4162

Source: SlideShare

Source: SlideShare

The way forward:

Investigating Neobeguea ankaranensis
N. ankaranensis

N. leandriana

N. mahafalensis

Investigating the mechanism(s)

of action of the active compounds.
Identifying targets

 Bringing hits/leads into drug
development

Networking
Patenting
Fundraising
Taking risks

Source: Nature 2008, 453, 840.

Acknowledgments

Oslo in October 2013

WIPO Award for young scientist

Funds from Uppsala Bio-X, Swedish VR, TWAS/UNESCO