Hydroxylation of yohimbine in superacidic media: One-step access to human metabolites 10 and 11-hydroxyyohimbine

Article abstract of DOI:10.1021/np000425z

Two major human metabolites of yohimbine (1), 10- and 11-hydroxyyohimbine (2 and 3), were prepared by direct hydroxylation of 1 under superacidic conditions. In this medium, the four positions of the benzene part of yohimbine were hydroxylated and the cor

Full text of DOI:10.1021/np000425z

J . Nat. Prod. 2001, 64, 193-195
193
Hyd r oxyla tion of Yoh im bin e in Su p er a cid ic Med ia : On e-Step Access to Hu m a n
Meta bolites 10 a n d 11-Hyd r oxyyoh im bin e
Alain Duflos,*,† Florence Redoules,† J acques Fahy,† J ean-Claude J acquesy,‡ and Marie-Paule J ouannetaud‡
Centre de Recherche Pierre Fabre, 17 Avenue J ean Moulin, Castres F 81106 Cedex, France, and Laboratoire de Synthe`se et
Re´activite´ des Substances Naturelles, UMR 6514, Universite´ de Poitiers, F 86022 Cedex, France
Received August 30, 2000
Two major human metabolites of yohimbine (1), 10- and 11-hydroxyyohimbine (2 and 3), were prepared
by direct hydroxylation of 1 under superacidic conditions. In this medium, the four positions of the benzene
part of yohimbine were hydroxylated and the corresponding monohydroxylated compounds (2-5) were
isolated. The structures of 2-5 were elucidated by spectroscopic methods including 1D and 2D NMR
techniques.
Yohimbine (1) is an indole alkaloid extracted principally
from the inner bark of the yohimbe tree, which grows wild
throughout Africa. Yohimbine is known as a potent and
selective R2 adrenoreceptor antagonist agent,1 but it does
not show selectivity among the three R2 adrenoreceptor
subtypes.2 Yohimbine is also well-known for its activity
against male impotence and, recently, against female
sexual disorders.3
the conditions, a simple mixture of monohydroxylated
yohimbine derivatives was obtained (Scheme 1), the four
positions of the benzene ring of yohimbine being hydroxy-
lated. The yield of each compound in the crude mixture
after hydrolysis of the superacidic medium was evaluated
by analytical HPLC. The mixture contained around 32%
10-hydroxyyohimbine (2), 23% 11-hydroxyyohimbine (3),
3% 12-hydroxyyohimbine (4), and 11% 9-hydroxyyohimbine
(5). Yohimbine (3%) remained, giving a total of 72%
identified compounds. Preparative chromatography, then
recrystallization, yielded pure compounds 2 and 3. Re-
peated chromatography yielded nearly pure compounds 4
and 5.
Mass spectral analyses of two human metabolites of
yohimbine (2 and 3), and comparison with authentic
samples, indicated that these were 10- and 11-hydroxy-
yohimbine.4 Compounds 2 and 3 were studied as selective
R2 adrenoreceptor antagonists,5,6 and the activity of 11-
hydroxyyohimbine (3) on these receptors has been pat-
ented.7
Yohimbine (1) and compounds 2-5 were studied under
identical conditions by various NMR techniques using
MeOH as solvent. The stereochemistry of the polycyclic
structure was studied by NOESY experimentation. The 1H
(Table 1) and 13C (Table 2) NMR chemical shifts were
assigned on the basis of 1D (1H, 13C, and DEPT) and 2D
(COSY, HMQC, and HMBC) spectra. Elucidation of the
position of hydroxylation was carried out using COSY and
HMBC spectra. For each aromatic carbon atom, the chemi-
cal shift variation between each hydroxyyohimbine deriva-
tive and yohimbine confirmed the assignments. Strong
NOE between the protons in position 6 and the proton in
position 9 for compounds 2, 3, and 4 provided further proof
permitting differentiation of compounds 4 and 5. For the
nonaromatic part of the yohimbine derivatives, analyses
of the differences of 13C and 1H chemical shifts between
each hydroxyyohimbine derivative and yohimbine con-
firmed that this part of yohimbine remained unchanged
(Tables 1 and 2). In addition, NOESY experiments showed
strong NOE between the axial protons on each face of the
nonaromatic polycyclic part of the yohimbine derivatives.
The biological hydroxylation of yohimbine by fungi has
been recognized for many years. In this way, Loo and
Reidenberg,8 Patterson et al.,9 and Hartmann et al.10
obtained 2 or 3. Adam et al.11 reported the NMR spectra
of 2. Concurrently, Levy et al. prepared authentic samples
of 2 and 3 by demethylation of methoxy compounds12
isolated from natural sources.13 Due to the scarcity of the
natural compounds and the poor yields of demethylation
reactions, we decided to reinvestigate the partial synthesis
of 2 and 3 by applying the unusual reactivity of superacids.
Resu lts a n d Discu ssion
The advantages of electrophilic hydroxylation of aromat-
ics with hydrogen peroxide in superacidic media were
described by Olah et al.14,15 We previously described the
electrophilic hydroxylation of indole derivatives by hydro-
gen peroxide under superacidic conditions.16,17 The oxidiz-
ing agent, source of “OH+”, reacted at the benzene ring on
the protonated indole principally at positions 5 and 6,
corresponding to positions 10 and 11 for yohimbine. It was
observed also that the more substituted indoles gave better
yields. Using the same strategy with 1 in the presence of
hydrogen peroxide, a mixture of hydroxylated derivatives
was obtained, together with a large quantity of tar. By
using sodium persulfate in place of hydrogen peroxide,
yohimbine hydrochloride in place of 1, and optimization of
It should be emphasized that the use of superacidic
media permitted oxidation of the aromatic ring without
modification of the remainder of yohimbine. In fact, pro-
tonation of all the functional groups present in the molecule
appears to protect fragile centers and to favor electrophilic
hydroxylation. The 17-hydroxy group remained intact,
without dehydration, and the configuration of the five
asymmetric centers was unchanged. This strategy provided
direct access to the two identified yohimbine metabolites
(2 and 3), in one step and with a relatively good yield, along
with two compounds (4 and 5) that have not been previ-
ously reported.
* To whom correspondence should be addressed. Tel: (33) 5 63 71 43
58. Fax: (33) 5 63 71 42 99. E-mail: alain.duflos@pierre-fabre.com.
† Centre de Recherche Pierre Fabre.
‡ Laboratoire de Synthe`se et Re´activite´ des Substances Naturelles.
10.1021/np000425z CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/18/2001

194 J ournal of Natural Products, 2001, Vol. 64, No. 2
Duflos et al.
Sch em e 1
Ta ble 1. 1H NMR Data (δ) of Compounds 1-5 in CD3OD
Avance 400 spectrometer. TMS was used as internal standard,
and samples were dissolved in CD3OD. Analytical HPLC
conditions: Merck LaChrom system with a photodiode array
detector (L 7455, L7100, L7200, D7000), Waters Symmetry
C18 5 µm (250 × 4.6 mm, ref: WAT 054215), gradient (0 min,
MeOH 10%, KCl 75 mM pH 2.5 with HCl (KCl) 82%, aceto-
nitrile (AN) 8%; 15 min, MeOH 20%, KCl 72%, AN 8%; 20 min,
MeOH 40%, KCl 52% AN 8%; 30 min, MeOH 40%, KCl 52%
AN 8%), flow 1 mL/min. Retention time: 10-hydroxyyohimbine
(2), 9.6 min; 11-hydroxyyohimbine (3), 10.9 min; 9-hydroxy-
yohimbine (5), 14.7 min; 12-hydroxyyohimbine (4), 16.3 min;
and yohimbine (1), 23.5 min. Yields in the crude mixture after
hydrolysis of the superacidic medium were estimated after
isolation and purification of each of the four hydroxylated
yohimbine derivatives. UV spectra of compounds 2, 3, 4, 5,
and yohimbine (1) differ significantly, and calibration of the
chromatogram was accomplished by an external standard
method using these five compounds as standards. Preparative
HPLC conditions: pump and detector system, Merck SepTech
(NovaPrep 5000); pre-packed column, RT Merck Hibar
(250 × 25, Lichrospher RP18, 5 µm); column, Prochrom (LC
50-VE); silica gel, Merck Si 60 (15-25 µm ref: 109336); and
reversed phase, C18 Lichroprep RP18 (15-25 µm ref: 113901).
Mass spectrometry data were obtained on a Finnigan MAT
TSQ 7000. For APCI, the temperature of the vaporizer and
capillary were respectively 400 and 150 °C. Corona current
was set to 5 µA. For DCI, the energy of the electron beam and
ion source temperature were respectively set to 70 eV and 150
°C, the pressure of the reagent gas (CH4) was 2400 mT, and
the desorption parameter was 50-500 mA in 0.2 min.
1
2
3
4
5
H-3R ax
H-5R ax
H-5â eq
H-6R eq
H-6â ax
H-9
H-10
H-11
H-12
H-14R eq
H-14â ax
H-15R ax
H-16â ax
H-17â eq
H-18R eq
H-18â ax
H-19R ax
H-19â eq
H-20â ax
H-21R ax
H-21â eq
H-23
3.3
2.6
3.0
2.7
3.0
7.4
7.0
7.0
7.3
2.4
1.1
2.0
2.3
4.2
1.9
1.6
1.5
1.4
1.5
2.2
2.9
3.8
3.3
2.5
3.0
2.9
2.6
6.8
3.4
2.7
3.1
2.7
3.0
7.2
6.6
3.4
2.6
3.1
2.7
3.0
6.9
6.8
6.5
3.3
2.6
3.0
3.2
3.0
6.3
6.8
6.8
2.4
1.2
2.0
2.3
4.2
1.9
1.6
1.5
1.4
1.5
2.2
2.9
3.8
6.6
7.1
2.4
1.1
1.9
2.3
4.2
1.9
1.6
1.5
1.3
1.5
2.1
2.8
3.8
6.8
2.5
1.2
2.0
2.4
4.3
2.0
1.7
1.6
1.4
1.6
2.3
3.0
3.8
2.5
1.2
2.0
2.4
4.2
1.9
1.7
1.5
1.4
1.5
2.3
2.9
3.8
Ta ble 2. 13C NMR Data (δ) of Compounds 1-5 in CD3OD
1
2
3
4
5
C-2
C-3
135.7
61.8
136.6
61.9
134.1
62.0
135.1
62.1
133.5
62.0
C-5
54.5
54.2
54.3
54.2
54.6
C-6
C-7
C-8
C-9
22.4
22.5
22.5
22.7
24.5
Hyd r oxyla tion of 1. Anhydrous hydrogen fluoride (200
mL) was introduced into a Teflon reactor and mechanically
stirred at -45 °C. Antimony pentafluoride (260 g) was cau-
tiously added over 3 min, and the temperature reached -10
°C. At -45 °C, sodium persulfate (7 g, 0.029 mol) was added.
Then, a solution of yohimbine hydrochloride (10 g, 0.025 mol)
in anhydrous hydrogen fluoride (60 mL) was introduced,
maintaining the temperature below -35 °C. This mixture was
stirred for 1 h and then, very carefully, poured into a vigorously
stirred mixture of Na2CO3 (500 g), H2O (500 mL), and ice
(2 kg). The aqueous phase was extracted three times with a
mixture of ethyl acetate/2-butanone (3:1, 3 × 100 mL). The
combined organic phase was washed three times with saline,
dried over Na2SO4, and evaporated to dryness. The residue
was dissolved and diluted to exactly 200 mL with MeOH. The
solution was chromatographed to determine the amounts of
remaining 1 and of each of the hydroxylated derivatives (2-
5). Merck Si 60 silica gel (75 g) was added to the rest of the
solution, and the solvent was evaporated to dryness.
107.8
128.4
118.6
119.8
121.9
112.1
138.1
34.7
37.5
53.8
68.7
33.5
24.4
41.2
62.3
175.4
52.2
107.2
129.1
103.2
151.2
111.6
112.5
133.0
34.6
37.4
53.8
68.7
33.5
24.4
41.1
62.3
175.4
52.2
107.6
122.5
119.0
109.7
153.8
97.9
139.2
34.8
37.5
53.9
68.7
33.5
24.5
108.5
130.5
110.6
120.6
107.1
144.4
127.7
34.8
37.5
54.0
68.8
33.6
24.5
41.3
62.4
175.6
52.3
107.6
118.0
152.5
104.4
122.5
104.4
140.2
34.7
37.5
53.9
68.7
33.5
24.5
41.2
62.4
175.4
52.2
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
41.3
62.4
175.4
52.2
Exp er im en ta l Section
Isola tion a n d P u r ifica tion . The crude mixture supported
on silica gel was introduced onto the top of the chromato-
graphic column (Prochrom LC 50-VE) packed with Merck Si
60 silica gel (250 g) and eluted (50 mL/min) with a gradient
from 100% CHCl3 to 90% CHCl3, 8% MeOH, 2% aqueous NH3
over 2 h. The fractions were selected in three groups after
analytical chromatography. The first group of fractions con-
tained principally 12-hydroxyyohimbine (4). The second group
of fractions contained a mixture of 9-hydroxyyohimbine (5)
(12%), 10-hydroxyyohimbine (2) (33%), and 11-hydroxyyohim-
bine (3) (55%). The third group of fractions contained a mixture
of 5 (3%), 2 (67%), and 3 (30%).
Gen er a l Exp er im en ta l P r oced u r es. Th e a u th or s d r a w
th e r ea d er s’ a tten tion to th e d a n ger ou s fea tu r es of
su per a cid ic ch em istr y. Ha n d lin g of h yd r ogen flu or id e
a n d a n tim on y pen ta flu or id e m u st be d on e by exper ien ced
ch em ists with a ll th e n ecessa r y sa fety a r r a n gem en ts in
pla ce.
Anhydrous hydrogen fluoride (Praxair), antimony penta-
fluoride (Merck ref: 812034), yohimbine (Sigma ref: Y3125),
and sodium persulfate (Degussa ref: EWG Nr. 231-892-1) were
used as received. 1H and 13C NMR, DEPT, COSY, HMQC,
HMBC, and NOESY were measured at 25 °C using a Bruker

Hydroxylation of Yohimbine
J ournal of Natural Products, 2001, Vol. 64, No. 2 195
12-Hyd r oxyyoh im bin e (4). The first group of fractions
was evaporated and the residue was subjected to reverse phase
chromatography using a prepacked column (250 × 25, Lichro-
spher RP18, 5 µm) eluted with a gradient from 95% A, 5% B
to 90% A, 10% B over 1 h (A: aqueous 1 M CH3COOH; B:
MeOH) to obtain 95% pure 4. The final purification was done
by chromatography with Merck Si 60 silica gel using a gradient
from 100% CHCl3 to 90% CHCl3, 8%, MeOH, 2% aqueous NH3
over 1 h to obtain 97.5% pure 5 (80 mg, 0.77% yield): white
C, 25% B (C: aqueous KCl 0.5 MeOH pH 2.8 with HCl; B:
MeOH) to obtain 95% pure 5. The final purification was done
by chromatography with Merck Si 60 silica gel and a gradient
from 100% CHCl3 to 90% CHCl3, 8% MeOH, 2% aqueous NH3
over 1 h to obtain 97.7% pure 5 (70 mg, 0.67% yield): white
amorphous solid; DCI+ m/z 371.1 [MH+]; 1H and 13C NMR,
Tables 1 and 2; anal. C 65.31%, H 7.88%, N 7.03%, calcd for
C
21H26N2O4, C 68.09%, H 7.07%, N 7.56%.
1
amorphous solid; APCI+ m/z 371.0 [MH+]; H and 13C NMR,
Ack n ow led gm en t. The authors thank Dr. J . P. Ribet and
Mr. P. Zalavari for their assistance in obtaining NMR, MS,
and elemental analysis data and Mr. J . C. Tristani for his
technical assistance.
Tables 1 and 2; anal. C 67.85%, H 7.18%, N 7.63%, calcd for
C
21H26N2O4, C 68.09%, H 7.07%, N 7.56%.
10-Hyd r oxyyoh im bin e (2). The third group of fractions
was evaporated, and the residue was subjected to crystalliza-
tion in MeOH to obtain 97% pure 3. Recrystallization using
2-butanone afforded 98.5% pure 3 (1.4 g, 13.5% yield): colorless
crystals; decomposition before melting; APCI+ m/z 371.3
Refer en ces a n d Notes
(1) Goldberg, M. R.; Robertson, D. Pharmacol. Rev. 1983, 35, 143-180.
(2) Ruffolo, R. R.; Bondinell, W.; Hieble, J . P. J . Med. Chem. 1995, 38,
3681-3716.
1
[MH+]; H and 13C NMR, Tables 1 and 2; anal. C 68.25.05%,
(3) Gorny, P. Patent WO9940917-A1, 1999.
H 7.27%, N 7.31%, calcd for C21H26N2O4, C 68.09%, H 7.07%,
N 7.56%.
(4) Le Verge, R.; Le Corre, P.; Chevanne, F.; Do¨e de Maindreville, M.;
Royer, D.; Levy, J . J . Chromatogr. Biomed. Appl. 1992, 574, 283-
292.
(5) Berlan, M.; Le Verge, R.; Galitzky, J .; Le Corre, P. Br. J . Pharmacol.
1993, 108, 927-932.
(6) Le Corre, P.; Peskind, E. R.; Chevanne, F.; Raskind, M. A.; Le Verge,
R. Eur. J . Clin. Pharmacol. 1997, 52, 135-138.
(7) Le Verge. Patent FR 2 686 881-A1, 1999.
11-Hyd r oxyyoh im bin e (3). The second group of fractions
was evaporated, and the residue was subjected to a second
chromatography with Merck Si 60 silica gel using a gradient
from 100% CHCl3 to 90% CHCl3, 8% MeOH, 2% aqueous NH3
over 1 h to obtain a mixture of 5 (3%), 2 (22%), and 3 (75%).
This mixture, crystallized with MeOH, afforded 95% pure 3.
The final purification was done by chromatography using a
prepacked column (250 × 25, Lichrospher RP18, 5 µm) eluted
with a gradient from 100% A, 0% B to 85% A, 15% B over 1 h,
then recrystallization using MeOH, affording 98% pure 3 (750
mg, 7% yield): colorless crystals; decomposition before melting;
APCI+ m/z 371.0 [MH+]; 1H and 13C NMR, Tables 1 and 2;
anal. C 65.31%, H 7.88%, N 7.03%, calcd for C21H26N2O4,CH3-
OH, C 65.65%, H 7.51%, N 6.96%.
(8) Loo, Y. H.; Reidenberg, M. Arch. Biochem. Biophys. 1959, 79, 257-
260.
(9) Patterson, E. L.; Andres, W. W.; Krause, E. F.; Hartman, R. E.;
Mitscher, L. A. Arch. Biochem. Biophys. 1963, 103, 117-123.
(10) Hartman, R. E.; Krause, E. F.; Andres, W. W.; Patterson, E. L. Appl.
Microbiol. 1964, 12, 138-140.
(11) Adam, J . M.; Fonzes, L.; Winternitz, F. Ann. Chim. 1973, 8, 71-78.
(12) Levy, J . Personal communication.
(13) Miet, C.; Croquelois, G.; Poisson, J . Phytochemistry 1977, 16, 803-
5.
(14) Olah, G. A.; Ohnishi, R. J . Org. Chem. 1978, 43, 865-7.
(15) Olah, G. A.; Fung, A. P.; Keumi, T. J . Org. Chem. 1981, 46, 4305-6.
(16) Berrier, C.; J acquesy, J . C.; J ouannetaud, M. P.; Renoux, A. New J .
Chem. 1987, 11, 611-615.
(17) Berrier, C.; J acquesy, J . C.; J ouannetaud, M. P.; Vidal, Y. Tetrahedron
1990, 46, 827-832.
9-Hyd r oxyyoh im bin e (5). A selection of chromatographic
fractions afforded a mixture of 5 (19%), 2 (44%), and 3 (37%).
This mixture was subjected to three reverse phase chromatog-
raphies using a prepacked column (250 × 25, Lichrospher
RP18, 5 µm) eluted with a gradient from 90% C, 10% B to 75%
NP000425Z