Regioselective acylation of 3,8-diamino-5-ethyl-6-phenyl-phenantridium bromide, preparation of potential new trypanocides

Termkdron Vol. 45, No. II. pp. 3385 to 3396. 1989 Printed in Great Britain.

0

034&4020/89 13.00-c .JO 1989 Pergamon Press

plc

REGIOSELECTIVEACYLATION OF 3,8-DIAMINO-5-ETHYL-6-PHENYL-PHENANTRIDIUM BROMIDE, PREPARATIONOF POTENTIAL NEWTRYPANOCIDES J. Laboratory

of

Loccufier

Organic

Krijgslaan

281 (S4), (Received

A methodology

was

trypanocide bromide,

the C-8

including tion of

the

allowed to

Direct

diacylation

improve

the

of

acylated, the

of

3,8-Diamino-5-ethyl-6-phenyl-phenantridium homidium) largely

(fig.1)

is

restricted

and to the

currently

to the study

use of

this

sequence

and

derivatives

the parent

bromide

compound in elucidating

the

A series

drug.

drug.

the pharmacokinetics

reoxida-

were prepared,

(ethidium

used as trypanocidal of

preferentially

in C-3 and C-8 position. of

the

(ethidium

a reaction

N-acylation

diacylated index

bromide

ring,

of

bromide

However,

aminogroups

respectively

therapeutical

modification

ethidium

phenantridium

the

1989)

regioselective

derivative.

Ghent

Belgium

phenantridium

acylation

of

University

Ghent,

in Belgium 22 February

allowing

amino substituted

reduction

C-8 and C-3,8

aiming

developed

State

B-9000

3,8-diamino-5-ethyl-6-phenyl homidium).

yielded

and E. Schacht*

Chemistry,

bromide data

Literature

or are

and pharmacodynamics, conformation

of

DNA and

either soluble

derivatives

RNA strings.

Fig.

1 : Ethidium

The scarce suitable The early

bromide

synthesis for

the

papers

study

were made

efforts of

by Walls

DNA and (5,

RNA,

to develop or

new water

6) and by Watkins

(7)

are,

new

pigments (l-4). to our knowledge,

the only reports describing the synthesis of chemical analogues or derivatives of ethidium bromide with the intention of obtaining compounds with improved pharmaceutical properties. 3385

J. LOCCLJRER and E. SCHACHT

3386

In this

paper

bromide with considerable

the

regioselectivity

S-ammonium function. (8,9)

(fig.21

a large

Direct

during

the acylation

of

ethidium

is reported. This regioselectivity is due to the of the C-3 aniline group by resonance with the Moreover, face to face dimerisation of ethidium bromide

has to be taken

extent

Fig.2

observed

amino acids desactivation

the whole

of

formed

ethidium

bromide

by ethidium

with

group.

= (CH,)cNH, =CH,

bromide

protected

butyloxycarbonyl

Scheme 1

= CH2

These two phenomena determine ethidium bromide.

dimer

shown in scheme 1. The tertiary amino protecting

account. of

: Face to face

acylation

into

chemistry

group

to

in solution.

amino acids occured (BOC) was selected

as a.8

Preparation of potential new trypanocides

3387

J. LOCCUFIER and E. SCHACHT

3388

The regioselectivity

of

scopy after acidolysis the NMR spectrum of

Starting from an interpretation of the BOC group. ethidium bromide, based on literature data (4,8),

spectra

of

aromatic

the

acylated

protons

differences

the parent

ethidium

Table

the

aromatic

for

shift

bromide

the

bromide

1 : Chemical

is

derivatives

governs

The shifts

the acylation

were analysed.

essential

ethidium

itself

summarized

are

and N-8-glycyl

for

on the

Given the

in table

protons (in

N-8-glycyl

Also

(A6 in ppm)

0.5% of

The analysis is

shift

+ 0.25

7.30

+ 0.02

6.37 7.26

+ 1.05 0.32

differences

minor differences

for

bromide

showed that

analysis of

proved

the

spectrum by the

ring

can

to ethidium

of The NMRspectrum remarkable anisotropy between

both

easily

of

(in

caused

From the

aniline

functions

are

acylated.

residual

was still

protons. assigned.

of

can

of

acylation

N-E-(BOC-gly-

ethidium

present

Bowever, The

DMSO) are given

by the

0.2ppm.

H1, H2 and H4, the

bromide

and

in the compound.

ethidium

N-3,8-ditryptophanyl

N-3,8-ditryptophanyl

CH protons it NMRdata

H7, Hg and HIO and

selectivity.

indol be

bromide

1% of

product the

the protons HPLC analysis

function.

below

the N-3,8-diacylated

for

the protons

on the C-8 aniline

complicated

phenantridium

shift

8.10

+ 0.74

elemental

relative

ethidium

+ 0.38

ethidium

acetate

D20).

7.20

chemical

to

ethidium

7.99

relatively

below

of

Ii9

the major

the

1.

trifluoroacetate

bromide

of

compared

HlO

must have occured cyl)

shift

of the

regioselectivity.

trifluoroacetate

(6 in ppm)

;;

NMR spectro-

The chemical

the aromatic

ethidium

ethidium

by proton

information

N-8-glycyl

differences

protons

investigated

ethidium

four

chemical

shift

in table 2. trifluoroacetate

6-phenyl group. The difference residues the two tryptophanyl be concluded

that

both

trifluoro-

protons

the

of

the

differences shows

a

in chemical around is C-3 and

C-8

3389

Preparation of potential new trypanocides

Table

2

the

Chemical

:

bromide

shift

differences

and N-3,8_ditryptophanyl aromatic

protons

for

ethidium ethidium

the

aromatic

protons

trifluoroacetate

bromide

(in

complex N-3,8

7.29

+ 1.11

Ii10

8.52

+

part

pattern,

by the acylation

with

group

(fig.

Fig.

ratio

N-3,8-dilysyl to

ethidium

a mixture

of

both

chloride

shows

a

and

a

of a N-8 acylated

reaction

products

is

80/20

from the NMR integrations. the

amino acid

accounted

ammonium function

N-acylated

to the

side

in position

the C-3 aniline

on the

Further

resulting

for

regioselectivity

of

5. This

group.

phenomenon described

derivatives,

chain

desactivation

the C-3

forms more,

a kinetic it

ethidium

of

restric-

can be anticipa-

bromide

in a shielding

aniline

can

also

the C-3-aniline

3).

3 : Shielding

of

the C-3 aniline

given

reaction

ethidium

bromide

Under the glycyl)

can be of

of

The of

the dimerisation

occur

spectrum

can be assigned

influence

function that

the

as calculated

tion ted

of

0.73

8

derivative.

reaction

for

ref.

which

acylated

the

0.87 0.72

H9

The remarkable of

+

7.48 8.58

: see

(N-3,8/N-8),

(Aa in ppm)

;:

* spectrum

ethidium

N-3,8_tryptophanyl

(6 in ppm)*

The aromatic

of

DHSO d8).

conditions, is

apparently

function the

by face

to face

concentration

so low that

the

of

dimerisation. free

reaction

N-8-(BOCvirtually

J. LOCCUFIERand E. SCHACHT

3390

stops

at

that

It

Intermolecular

stage.

regioselectivity tine.

of

the

can be anticipated

acylation

that

increases

the

sterical

a result,

the

C-3 aniline

interactions

reaction

increasing

hindrance

are

ethidium

bulkiness

of

becomes

r5.l f&g

responsible bromide

the

and hampers the dimer

function

for

with

amino acid formation

more susceptible

for

the

BOC-gly-

side

chain

(fig.

4).

As

reaction.

--

0

0

of

oz,,,...

l-INN

$0

H

k

This

4 : Sterical

hypothesis

di-BOC-lysine, having side of

is

chain

but

accessible

a bulky rigid

apparently Bence, and the

For the preparation

in of

has to be made fully that

sodium borohydride

tion

into

Fig.

results

causes latter

goes

reduced

amino acid obtained

side

chain,

during

case

of

to some extent,

much larger the

side

and with

The flexibility

sterical

C-3 aniline

the while

chains. coupling

with

BOC tryptophan di-BOC-lysine the

rigidity

restrictions function

for

is

fully

to completion.

the N-3,8-diglycyl

accessible.

the DNA double

by the

dimerisation

the

reaction

by bulky

chain.

chain

)NH2

caused

but flexible

side

allows

side

tryptophan

dimerisation.

substantiated

having

a bulky

the

hindrance

0 “i/

0

I N

Fig.

oj

It

derivative

was demonstrated

ethidium

bromide

the C-3 aniline

no longer

showed intercalla-

helix.

5 : Sodium borohydride

reduced

function

by Thomas and Roques

ethidium

bromide.

(8)

Preparation of potential new trypanocides

It

was assumed

Given

the

must

provide

that

reduced

reoxidation

derivatives

of

us with

reduction,

the salts

has

ethidium

the

described

was applied

bromide

approach

on both

method

The reoxidation

reduced

a handy

acylated

quinolium

ethidium

to

lost

bromide

the

its is

ability

by Elderfield

to dimerize.

feasible,

preparation

and C-8

C-3

3391

of

aniline

the

bromide

functions.

and Wark for

latter

ethidium the

For

the

reduction

of

(10).

to N-3,8-di(BOC

glycyl)

ethidium

iodide

is

shown

in scheme

2.

Scheme 2

I2 - TEA HcOH

HOAc-HBr -

The intermediates The chemical

range

not

isolated

because

of

of

the

aromatic

protons

are

shifts

The NWRspectrum long

were

phenyl

derable

downfield

occured

on both

of

N-3,8-di(BOC

anisotropy shift the

C-3

of

its

data ability

amino

substantiate to

functions.

dimerize

all

glycyl) b

for

our

out

with

hypothesis

and provides

light

given

ethidium

the

aromatic

and C-8 aniline

that reoxidation was carried elemental analysis. These

(

their

protons

that

reduced

the

possibility

that

for

remarkable The

consi-

acylation

was further ethidium

a

ppm).

The NWRdata which

sensitivity

3.

shows

: 0.05

indicates

function. success,

in table

iodide

BGC groups

and air

further

has prove

confirmed

bromide reaction

by

has

lost

on

both

J. Loccunrx and E.

3392 Table

3 : Chemical

shift

ethidium the

aromatic

N-3,8-di(BOC

bromide

glycyl)

(DMSO d6).

N-3,8-di(BOC

glycyl)

7.48 8.58

+ 0.57 0.77

;;

6.18 7.32

+ 1.77 1.78

H9

7.29

+ 1.16

HlO

8.52

+ 0.53

functions

the

feasibility of

was

for

ethidium

attributed

dimerixation amino acid

sufficiently

ethidium

of

bromide

,“:

work,

bulky

protons

protons

to ethidium

(A6 in ppm)

In this

face

the aromatic

compared

(8 in ppm)

aniline selectivity

of

iodide

SCHACHT

to

of

ethidium

side

chains

as to

achieve

selective

bromide

has

acylation

intermolecular bromide

of

the

C-3 and

been demonstrated. interactions,

derivatives.

proved

to hamper the

quantitative

acylation

The

namely face

to

hindrance

by

Sterical formation on both

C-8

regio-

of

the

the

dimers

C-3 and

C-8

amino function. By reduction dimerize,

of

the phenantridium

which

reoxidation

of

biological

NMR spectra

aniline

reduced

evaluation

forthcoming

Materials

makes both the

of

ring , ethidium functions

intermediates the

ethidium

bromide

loses

available

was carried derivatives

its

to

reaction.

for out with

will

ability

be

The

success.

The

discussed

in

a

publication.

: were

recorded

on a 200 MHz (WP-Bruker)

NMR spectrometer.

TLC analyses

The HPLC analysis

was effected

were on

and a 360 MHz

run on Merck

a lo,

(WP-Bruker)

Kieselgel

60 F254

Waters

C-18 column,

Boundapack

plates. with

W detection. NMR analysis of ethidium bromide : 360 MHz 'H NMR spectrum of ethidium bromide in D20 : 6=1.3, m=3 (38); the methylene the S-ethyl group group

: 6~4.43,

m=2x2, 52-1~8.8

m=4 (2H);

H1 : 6~8.10,

Hz, 52-4’2

Hz (1H) : 84 : 6=7.26,

: the methyl protons

m=2, 51-2’8.8 J4_2=2

Bz (1B);

of

protons

of

the

S-ethyl

Ii2 :

6=7.30,

Hz; H7 : 636.37,

m=2,

3393

Preparationof potentialnewtrypanocides ~7-9’2

Hz (1s);

bs7.99, group

Hg : 6=7.20,

m=2, Jlo_g=8.8

of

To a solution

of

N-8-glycyl

The reaction

and protected

is

allowed is

orange

an

The product

is

(eluent

MeOH/NH3 (0.5%))

200 MHz ‘H

NNR data

the

methylene

protons

Hl : 6~8.86, 6~7.86,

(DMSO d6)

the methyl

the methylene

aniline

I$’

:

C-phenyl group :

protons

protons protons

: 1=6.77,

m (6H);

of of

of

the

24 hours

is

orange group

separated

of

urethane

Hg : i3=8.38,

: 6=3.81,

: 5=7.10,

m=2

the m=3

6-phenyl

m=2 (1H);

:

(together

m-4 (28);

H4 + the

the dried

B0C groups m=3

chain

proton

and

Rf’0.3.

the

: h-4.62,

50

storage from

ether

: h-1.44,

side

group

at O°C

pressure.

48 hours

spot

protons

m=2 (1H);

m=l (1H);

reduced

, washed with

5-ethyl

the

mmol) DMAP are

under nitrogen

After

product

methyl

5-ethyl

H* : 6=7.52,

H7 : 6= 7.57,

residue.

the BOC-glycine

the

m=l (2H);

m=2 (1H);

for

: a bright

: the

in 15 ml dry DMF, 270 mg

removed under

by filtration

TLC analysis m=l;

is

cristalline

isolated

70%).

(2H);

(1s);

:

bromide

added to the oily

under vacuum (yield:

12 H);

Hz

DCC and 60 mg (0.49

to continue

The solvent

(l/3)

refrigerator,

b=l.38,

, J9_lo=8.8

trifluoroacetate

mmol) ethidium

260 mg (1.26mmol)

from light.

ml methanol/ether in the

ethidium

250 mg (0.63

(126 mmol) BOC-glycine,

solution.

Hz

m (2H).

The preparation

added.

J9_7=2

(1H); the ortho and para protons of the the meta protons of the 6-phenyl m (3H);

&=7.70-7.80,

:

6=7.26-7.30,

m-2x2,

HZ

C-3 (1H);

group

H1’ :

:

b=8.95,

m=2 (1H). Elemental

analysis

: C28H3103N4Br (551.48)

HPLC analysis lop time

(eluent

Boundapack 4 min.

Impurities

Waters

C-18

200 mg (0.36

acid.

filtration,

The

60.98%

5.67%

10.15%

61.05%

5.83%

9.98%

(80/20)

was allowed

360 MHz 1H NNR data m=2, J2_l=8.9 m=2,

of

the

bromide

mixture to stand

ether

(D20)

Hz (1H); Jg_lo=9

(elution

0.03% HC104, column at 290nm) :

ethidfum

bromide

is

C-phenyl

over

: b=8.35,

H4 : W7.58, Hz (1H);

: 4 min. time

and dried

: El

time

(elution

in 200 ml ether,

washed with

meta protons

N

cm, W detection

N-8-(BOC-glycyl)

precipitated

The mixture

bromide

ethidium

mmol) of

trifluoroacetic

b17.94,

(30 x 0.5

H

: elution

25 sec. : < 1% ethidium

is

found : acetonitrile/water

N-3,8-di(B0C-glycyl)

product

C expected

m=l

stirred

for

night.

is

m=2, Jl_2=8.9 8’

and

dissolved

hours.

The is

all

t 6=7.42,

ml

reaction

isolated

yield

Bz (1H);

in 5

residue.

a cristalline (over

<0.5%

12 sec.)

The residue

under vacuum (1H);

: 3 min.

3

which yields

50 sec.)

by

60%).

Hz :

bs7.32,

m=l (la);

Hg

RlO : br8.37, m=2, J10_g=9Hz (1H); the para group : 6=7.8-7.9, m (3H); the ortho protons

:

and of

J.

3394

the

6-phenyl m=l;

b=4,

group

:

the CE2

Of

the CA3 of

of

To a solution

excess

of

of

is

ting

group

carried

intense

the

intermediate reaction

precipitated

products, was

in ether.

was dried

in vacuum.

The aromatic

for

Rf=0.8

After

to

36 hours.

The isolated

system

of

namely N-3,8-dilysyl

5 ml

of

for

product

bromide

orange

acetic

2

added.

with

was analysed

The at showed

spot

Rf’0.75 isola-

without acid

hours.

satured

The

with

product

ether,

the

was

product

by NMR spectroscopy.

showed the presence

and N-8-lysyl

large

TLC analysis

minor

and washing

the ‘H NMR spectrum

ethidium

(28) t

from light,

were hydrolysed

continue

filtration

DMAP are

protected

and a

by adding

allowed

:

HZ

in 2 ml dry DMF a

amount of

atmosphere,

continue

spot

substituent

:

bromide

0.5% NH3). The BOC groups

: methanol/

HCl. The

yellow

chloride

catalytic

nitrogen to

N-8-glycyl

m=4, JCH2_~~3’7.3

m=3, JCH2_CH3=7.3 Hz (3H).

ethidium

DCC and a

out under

CH2 of

b=4.63,

ethidium mmol)

was allowed

one

the

group

: b=l.46,

N-3,8-dilysyl

50 mg (0.13

O°C. The reaction two spots,

m=2 (2H);

N-S-ethyl

diBOC lysine,

reaction

(eluent

8~7.52, the

the N-S-ethyl

The preparation

LOCCUFIERand E. SCHACHT

ethidium

of

two products

bromide

(yield

:

70%). 360 MHz lH NMR data and b-CH2

of

and b=1.9; b=1.9 the

lysine

a-CA of

br8.08,

the

group

Ii9

(N-3,8) (solvent

(N-3,8)

(~-3~8)

:

: b=9.08,

440 mg (1.45

ethidium

and N-8)

:

system

(N-3,8)

bt9.13,

:

(N-8) m=2;

-

b=1.68 between

and b-3.05,

m=3;

m ; the CA2 of

the

I b=7.58,

multiplet;

and H2

(N-8)

H1’

between

II2 (N-8)

complex

m=3r the

multiplet

: a=3

chain

b=7.79,

I$ and

multiplets

m and b4.35,

m=2; Hg

m=4;

of

light

bromide

ethidium

(20% based

bromide

I-3,8-ditryptophanyl

m=4;

H7 (N-8)

(N-3,8)

:

:

b=8.43,

: bt8.88,

1~2x2;

H4 (N-3,8)

: 9.18,

I$’ m=2

and under

isolating

the

trifluoro

acetic

yielding

is

allowed

inert

intermediate acid. a yellow

to

atmosphere. 2 hours

cristalline

washed several

times

(80% based

100 mg (0.25

on NMR integration).

reaction

stirring ether

products,

the product

precipitate. with

mmol) ethidium

The product

and dried

6 ml of

precipitated is

isolated

under vacuum

in

protected without

by adding is

(0.41

bromide

continue for 20 hours at OOC, The BOC groups are hydrolyzed

BOC protected

After

on NMR integration)

ethidium trifluoroacetate : 298 mg (1.45 mmol) DCC and 50 mg of

added to a solution

dry DMF. The reaction

filtration,

aromatic

t b18.25,

m=2; H1

side

: b=4.15,

: bnl.58,

: complex

chain

lysine

mmol) BOC-tryptophane,

mmol) DMAP is

ether

chain

complex

side

m=4; the

bs8.55,

= N-3,8-dilysyl

The preparation

from

the

group

MeOH d3).

= N-8-1~~~1

(N-3,8)

(N-3,8

: three

lysine of

side

: bs4.75,

the N-S-ethyl

chain

the

-CB2

lysine

m=2; H7

m=2x4;

(N-8)

the

group

C-phenyl

side

9-CIi2 of

and b=2.3;

N-S-ethyl the

the

the

the CH3 of

:

(yield

in by :

Preparation of potential new trypanocides

3395

75%). MHz ‘I3 NMR data (DMSO d6) : the CH3 of the N-S-ethyl group : 6~1.6, m=3 (3H); the CH of the N-8-tryptophanylsubstituent : 614.3, m--3 (la); the CH of the N-3-tryptophanylsubstituent : 6=4.5, m-3 (1H); the CH2 of the N-S-ethyl group : 6=4.7, m=4 (28); El: 6B9.30, m=2; H2 : 6~8.35, m=2; H4 : 639.05, m=l; 8' : 638.15, m=l; Hg : 6=8.40, m=2; H1' : 6=9.25, m=2; the 6-phenyl group : 6t7.9, m; the indol protons : H2' : 6=7.30 and aa7.40, m-l; H4' : 6~7.88 and 6=7.98, m=2; H5' : 657, m=3; H6' : 6=7.15, m=3; 87' : 6=7.42, m=2.

200

The preparation of N-3,8-diglycyl ethidium bromide : The preparation of reduced ethidium bromide : 200 mg (0.50 mmol) of ethidium bromide is dissolved in 6 ml absolute methanol, 88 mg NaBH4 is added at 0°C. The reaction is carried out under nitrogen, in absence of light and at O°C for 24 hours. The solvent is evaporated under reduced pressure and 6 ml of a 3% NaOH solution is added. The solution is extracted 6 times with 20 ml ether. The ether fractions are pooled and dried over anhydrous Na2S04. Ether is evaporated under reduced pressure and a light brown cristalline residue is obtained which is recristallixedfrom ether. For spectroscopical and conformationaldata see reference 8. The coupling of BOC glycine to reduced ethidium : To a solution of 250 mg (0.79 mmol) of reduced ethidium in 4 ml dry acetonitrile, a 5 fold excess of BOC-glycine and DCC is added to obtain a 100% acylation of the amino functions. The reaction must be carried out in the dark and under nitrogen due to the instability of reduced phenantridium molecules. The reaction is allowed to continue for 20 hours. After 20 hours the DCU formed is removed by filtration and the acetonitrile is removed in vacuum. Separation at this stage is not necessary. Reoxidation of N-3,8-di(BOC glycyllamino-6-ethyl-5-phenyl-5,6-dihydrophenantridine : The crude reduced product is dissolved in 20 ml methanol. 0.3 ml triethylamine and a slight excess of I2 (206mg, 0.8lmmol)are added. The mixture is refluxed until the solution becomes orange. The solution is concentrated to 5 ml and the product is precipitated in ether. .Redissolving in methanol, followed by precipitation in ether is used to purify the product (yield: 60%), 200 MHz 'H NMR data (DMSO d6) : the BOC groups : 651.4, m=l, 6~1.45, m=l; the CH3 of the N-S-ethyl group: 6r1.55, m=3 (together 21H)t the CH2 of the N-E-BOC-glycyl group : 6=3.7, m=2 (2H); the CR2 of the N-3-BOC-glycylgroup : 6z3.9, m=2 (2H); the CH2 of the N-S-ethyl group : 6~4.7, m=2 (ZH); the urethane proton of the N-B-BOC-glycyl group : 617.05, m=3 (1H); the urethane

3396

J.LOCCUFIER andE.SCHACHT

proton of the N-3-BGC-glycylgroup : b-7.2, m=3 (1H); H1 : b=9.15, m=2 (1H): m=l (1B); Hg : Ii2: b=8.25, m=2 (1H); II4: b=9.1, m=l (1H); B7 : b=7.9!?, b=8.45, m=2 (1H); I$' : bs9.05, m=2 (1A); the 6-phenyl group : b=7.85, m (5H) elemental analysis : C35H4206N51 H C N (755.65) expected 55.63% 5.60% 9.27% found 5.70% 55.84% 9.15% TLC analysis (eluent MeOH/NH3 0.5%) : a bright yellow spot Rf'0.7 The hydrolysis of the BQC groups : N-3,8-di(BOC glycyl)ethidiumiodide is dissolved in 5 ml acetic acid satured with HBr. After 2 hours the product is precipitated in ether, which resulted in a yellow cristalline product. (overall yield : 60%) 200 MHz 'H NMR data (DMSO d6) : the CH3 of the N-5-ethyl group : 811.51, m=3, JCH2_CH3'7 Hz (3H); the CH2 Of the N-8-glycyl group : b=3.8, m=l (ZH); the CH2 of the N-3-glycyl group : b=4.0, m=l (2H); the CR2 of the N-5-ethyl group : b-4.65, m=4 (2H); A1 : b=9.21, m=2, JHl_H2'10 Hz (1H); E2 : b18.28, m=4, JH2_Hl=lO Hz, JH2_84'2 Ax (1H); H4 : bs9.03, m=2, JH4_B2=2 Hx (1H); H7 t b=8.11, m=2, JH7_Hg=2.5 xz (1H)t Hg : 608.35, m=4, JHg_B10=9.2 Hz, +g_87’ 2.5Hz (IH); Alo : b-9.13, m=2, JH10_Hg=9.2Hz (1H); the C-phenyl group : b57.82, m (5H)

The authors wish to thank the "NatiOnaal Fonds voor WetenschappelijkOnderxoek for the research grant 3.0064.87 and for providing a research mandate to Johan Loccufier.

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