Absolute configuration of myrobotinol, new fused-hexacyclic alkaloid skeleton from Myrioneuron nutans

Article abstract of DOI:10.1021/jo7017203

(Chemical Equation Presented) Myrobotinol (1) was isolated from the leaves of Myrioneuron nutans (Rubiaceae) and its structure determined from spectral data, including mass spectrometry and 2D NMR. This compound presents a new hexacyclic alkaloid skeleton including a 1,3-oxazine and aminal functionality. The absolute configuration of myrobotinol was established by using Mosher's method. A plausible biosynthetic pathway starting from L-lysine via Δ-piperideine was proposed for this hexacyclic alkaloid.

Full text of DOI:10.1021/jo7017203

for myrobotinol (1) by condensation of two cis-DHQ derivatives,
Absolute Configuration of Myrobotinol, New
Fused-Hexacyclic Alkaloid Skeleton from
Myrioneuron nutans
biogenetically derived from L-lysine.2a,b
Van Cuong Pham,*,†,§ Akino Jossang,† Thierry Se´venet,‡
Van Hung Nguyen,§ and Bernard Bodo*,†
Laboratoire de Chimie et Biochimie des Substances
Naturelles-CNRS, Muse´um National d’Histoire Naturelle, 63 rue
Buffon, 75005 Paris, France, Institut de Chimie des Substances
Naturelles, CNRS-91198 Gif-sur-YVette cedex, France, and
Institute of Chemistry-Vietnamese Academy of Science and
Technology, 18 Hoang Quoc Viet road, Hanoi, Vietnam
The crude alkaloid fraction (29.5 g) obtained from the dry
leaves of M. nutans (5.5 kg) was purified by repeated open
column chromatography over silica gel eluted with CH2Cl2/
MeOH gradient to afford myrobotinol (1, 20 mg).
Myrobotinol (1) was obtained as an optically active colorless
crystalline solid, mp 225-226 °C, [R]20D +36.4 (c 0.4, MeOH).
In its ESI-MS, the protonated molecular ion [M + H]+ was
observed at m/z 333.2538 (calcd 333.2542 for C20H33N2O2), in
agreement with the molecular formula C20H32N2O2. The 1D
NMR (1H and 13C) spectra revealed nine sp3 methine and eleven
methylene groups. The six degrees of unsaturation were thus
assigned to six rings. 1H-1H COSY data allowed the determi-
nation of two sets of connections as indicated by bold bonds in
Figure 1. The A-fragment was defined through a set of
correlations extending from CH2-2 (δH 2.66 and 2.76) through
H-11 (δH 4.13) observed in the COSY 45 and TOCSY spectra.
In addition, H-10 (δH 3.11) was correlated to both H-5 (δH 1.92)
and H-9 (δH 2.24). Similarly, the B-fragment was determined
by correlations starting from CH2-13 (δH 3.25 and 3.98) to H-21
(δH 4.19) together with those of H-23 (δH 2.69) with H-14 (δH
2.15) and H-18 (δH 1.93). The chemical shifts of CH2-2 (δC
38.0, δH 2.66 and 2.76), CH-10 (δC 57.4, δH 3.11), and CH-23
(δC 64.5, δH 2.69) suggested they were attached to a nitrogen
and those of CH-11 (δC 91.6, δH 4.13) were characteristic of a
methine linked to both a nitrogen and an oxygen. In the HMBC
spectrum, the correlations of H-10 with C-2 and C-21 indicated
direct linkage of N-1 with C-2, C-10, and C-21. Furthermore,
H-11 was correlated to C-13, C-21, and C-23, this latter carbon
being further correlated to H-21 and CH2-13. From these data,
N-22 was linked to CH-11, CH-21, and CH-23 and in addition
both CH-11 and CH2-13 were bonded to O-12. The planar
structure of myrobotinol (1) was thus elucidated as drawn in
Figure 1.
bodo@mnhn.fr; phamVc@ich.Vast.ac.Vn
ReceiVed August 9, 2007
Myrobotinol (1) was isolated from the leaves of Myrioneuron
nutans (Rubiaceae) and its structure determined from spectral
data, including mass spectrometry and 2D NMR. This
compound presents a new hexacyclic alkaloid skeleton
including a 1,3-oxazine and aminal functionality. The
absolute configuration of myrobotinol was established by
using Mosher’s method. A plausible biosynthetic pathway
starting from L-lysine via ∆-piperideine was proposed for
this hexacyclic alkaloid.
The genus Myrioneuron produces a group of polyheterocyclic
alkaloids and we have previously reported the structural
elucidation and the total synthesis of several tricyclic alkaloids
from M. nutans.1a-c These compounds have various alkaloid
skeletons which all contain a cis-decahydroquinoline (cis-DHQ)
moiety and for some of them, an oxazine motif. In continuation
of our research of alkaloids from this plant, further purification
of the crude alkaloid fraction has led to the isolation of a new
hexacyclic alkaloid skeleton, which we designate as myrobotinol
(1). Its absolute configuration was determined by Mosher’s
method taking advantage of the presence of a secondary alcohol
function. Finally, a plausible biosynthetic pathway is described
FIGURE 1. 1H-1H COSY (;) and HMBC (f) correlations for 1.
The relative configuration of myrobotinol (1) was established
by analysis of 1H-1H coupling constants and NOE interactions.
Hence, the H-16 proton had two trans-diaxial (J1 ) J2 ) 11.3
Hz) and two gauche (J3 ) J4 ) 4.5 Hz) coupling constants
indicating its axial disposition on the a-ring. The five protons
† Muse´um National d’Histoire Naturelle.
‡ Institut de Chimie des Substances Naturelles.
§ Institute of Chemistry-Vietnamese Academy of Science and Technology.
(1) (a) Pham, V. C.; Jossang, A.; Chiaroni, A.; Se´venet, T.; Bodo, B.
Tetrahedron Lett. 2002, 43, 7565-7568. (b) Pham, V. C.; Jossang, A.;
Chiaroni, A.; Se´venet, T.; Nguyen, V. H.; Bodo, B. Org. Lett. 2007, 9,
3531. (c) Pham, V. C.; Jossang, A.; Se´venet, T.; Nguyen, V. H.; Bodo, B.
Tetrahedron 2007, 63, 11244.
(2) (a) Golebiewski, W. M.; Spenser, I. D. J. Am. Chem. Soc. 1984, 106,
7925. (b) Wanner, M. J.; Koomen, G. J. Stud. Nat. Prod. Chem. 1994, 14,
731.
10.1021/jo7017203 CCC: $37.00 © 2007 American Chemical Society
Published on Web 11/14/2007
9826
J. Org. Chem. 2007, 72, 9826-9829

TABLE 1. NMR Data for 1a
C no.
2
δC
δ
H m (J, Hz)
C no.
13
δC
δH m (J, Hz)
38.0
2.76 br dd (11.5, 4.6)
73.0
3.98 dd (10.9, 4.6)
2.66 br dd (11.5, 11.3, 2.6)
3.25 dd (10.9, 10.9)
3
4
25.9
24.0
1.71 m
1.49 m
1.53 m
14
15
26.6
37.0
2.15 ddddd (11.3, 11.3, 10.9, 4.6, 3.5)
1.68 m
1.27 m
1.92 m
1.48 m
1.48 m
0.79 ddd (11.3, 11.3, 11.3)
3.82 dddd (11.3, 11.3, 4.5, 4.5)
1.88 ddd (12.9, 4.5, 2.5)
1.45 m
5
6
34.9
31.2
16
17
65.6
40.0
7
8
9
20.1
27.4
30.6
1.38 m
1.38 m
1.40 m
1.32 m
18
19
20
36.1
25.3
30.0
1.93 m
1.61 m
1.51 m
1.65 m
2.24 dddd (11.9, 11.9, 3.5, 3.5)
1.65 m
10
11
57.4
91.6
3.11 dd (11.9, 4.8)
4.13 d (3.5)
21
23
68.7
64.5
4.19 dd (9.3, 3.9)
2.69 dd (11.3, 4.5)
1
a CDCl3, 298 K; H 400.13 MHz, 13C 75.47 MHz.
to which H-14 was coupled were determined from the COSY
spectrum as usual and four of them gave very well resolved
and isolated signals from which it was easy to determine the
reciprocal values of the coupling constants. These values were
confirmed by the analysis of the aspect of the cross-peaks of
the COSY and HSQC spectra, which in addition allowed the
determination of the last coupling constant.3 The H-14 proton
presented five coupling constants: three large characteristic of
trans-diaxial relationships (J1 ) 11.3 Hz, J2 ) 11.3 Hz, and J3
) 10.9 Hz) and two gauche (J4 ) 4.6 Hz and J5 ) 3.5 Hz).
This indicated that H-14 was axial on both the a- and c-rings
and that it was in trans-diaxial relationship with H-23 which,
in turn, appeared as a doublet of doublet with a trans-diaxial
(J1 ) 11.3 Hz) disposition and a small (J2 ) 4.5 Hz) coupling
constant: H-23 was thus in a gauche relationship with H-18.
Furthermore, as the mutual coupling between H-11 and H-9
was small (J ) 3.5 Hz) they were in a cis relationship. H-10
was in the trans-diaxial disposition with H-9 on both the d-
and f-rings, as signified by their mutual coupling constant (J )
11.9 Hz). However, H-10 appeared as a double doublet (J1 )
11.9, J2 ) 4.8 Hz) thus depicting its cis relationship with H-5.
Finally, H-21 had a large (J1 ) 9.3 Hz) and a small (J2 ) 3.9
Hz) coupling constant and was thus axial on the b-ring. These
results were confirmed from NOEs data obtained by a NOESY
experiment: spatial interactions of H-23 with H-18, H-17ax,
H-15ax, H-13ax, and H-11, as well as those of H-21 with H-19ax,
H-14, and H-10, were observed (Figure 2). H-9 was also
correlated with H-7ax, H-4ax, and H-2ax. The correlations between
H-10 and both H-14 and H-21 were presented. Finaly, H-16
was correlated with H-14. Complete analysis permitted estab-
lishing the relative configuration for myrobotinol (1) as 5S*,9R*,-
10R*,11R*,14R*,16R*,18R*,21R*,23R* (Figure 2), in which
two cis-DHQ moieties substituted at C-8 were linked to each
other via 1,3-oxazine and aminal functionality.
FIGURE 2. Selected NOE interactions for 1.
(R)- and (S)-MTPA-myrobotinol esters 2 and 3 were prepared
by esterification of myrobotinol (1) with (R)- and (S)-MTPA,
respectively. The structures of the esters 2 and 3 were assigned
by 2D NMR. The differences of proton chemical shifts between
esters 2 and 3, ∆δR-S (δ(R)-MTPA-myrobotinol - δ(S)-MTPA-myrobotinol
)
were calculated (Figure 3). These values were positive on the
up side of the molecule 1, while they were negative on the other
side. By using Mosher’s model, the absolute configuration at
the chiral carbon C-16 was thus determined as R (Figure 3).
According to the relative configuration established above, the
absolute configuration of myrobotinol (1) was defined as 5S,9R,-
10R,11R,14R,16R,18R,21R,23R. This compound is a new
alkaloid skeleton with six fused rings and named myrobotinol.
Retrosynthesis of myrobotinol (1) suggests that it could be
biosynthesized from condensation of the two related cis-DHQ
fragments 4 and 5, followed by cyclization (Scheme 1).
According to this hypothesis, the intermediates 4 and 5 resulting
from L-lysine via ∆-piperideine2a,b are condensed to afford
(4) (a) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34,
2543. (b) Sullivan, G. R.; Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc.
1973, 38, 2143. (c) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95,
512. (d) Trost, B. M.; Belletire, J. L.; Godleski, S.; McDougal, P. G.;
Balkovec, J. M. J. Org. Chem. 1986, 51, 2370. (e) Latypov, S. K.; Seco, J.
M.; Quinoa, E.; Riguera, R. J. Org. Chem. 1996, 61, 8569. (f) Seco, J. M.;
Latypov, S. K.; Quinoa, E.; Riguera, R. J. Org. Chem. 1997, 62, 7569. (g)
Xiao, L.; Yamazaki, T.; Kitazume, T.; Yonezawa, T.; Sakamoto, Y.;
Nogawa, K. J. Fluorine Chem. 1997, 84, 19. (h) Takahashi, H.; Iwashima,
M.; Iguchi, K. Tetrahedron Lett. 1999, 40, 333. (i) Porto, S.; Dura´n, J.;
Seco, J. M.; Quin˜oa´, E.; Riguera, R. Org. Lett. 2003, 5, 2979. (j) Zhang,
Q. Y.; Carrera, G.; Gomes, M. J. S.; Aires-de-Sousa, A. J. Org. Chem.
2005, 70, 2120.
The absolute configuration of chiral secondary alcohols can
be determined from NMR analysis of their derivatives with
chiral auxiliary reagents such as methoxytrifluoromethylphe-
nylacetic acid (MTPA) or methoxyphenylacetic acid (MPA).4a-j
According to the former strategy, the absolute configuration of
1 was determined by Mosher’s method, taking advantage of
the presence of the secondary alcohol function at C-16. The
(3) Simova, S. Magn. Reson. Chem. 1998, 36, 505.
J. Org. Chem, Vol. 72, No. 25, 2007 9827

1
1
FIGURE 3. (A) H NMR (400.13 MHz, CDCl3, 298 K) of (R)-MTPA-myrobotinol (2); (B) H NMR of (S)-MTPA-myrobotinol (3).
SCHEME 1. Proposed Biosynthetic Pathway for Myrobotinol (1)
compound 6. Oxidation of 6 provides the imine 7, which is
further cyclized to complete the biosynthesis of myrobotinol
(1) (Scheme 1). The presence of myrioxazines A and B1a in the
plant M. nutans provides support for the hypothetical biosyn-
thetic pathway shown in Scheme 1 for myrobotinol (1).
Cytotoxic and antiplasmodial activities of myrobotinol (1)
were evaluated on KB cell lines and on Plasmodium falciparum
(FcB1 strain), respectively. Myrobotinol (1) was found to be
inactive in two cases (IC50: >33 µg/mL on KB and 20 µg/mL
(56 µM) on P. falciparum). However, its MTPA derivatives 2
and 3 showed significant activity on KB cell lines and on P.
falciparum. For cytotoxicity on KB cell, 2 and 3 displayed an
IC50 of 2 µg/mL (4.5 µM) and 3 µg/mL (6 µM), respectively,
while these values were 2 µg/mL (4.5 µM) for both 2 and 3
when evaluated on P. falciparum. This observation suggested
that the antiplasmodial activity of 2 and 3 could be due to their
cytotoxicity.
In summary, myrobotinol (1), a novel alkaloid with an
unprecedented fused-hexacyclic skeleton, was isolated from M.
nutans. The absolute configuration of 1 was established by using
Mosher’s method. For understanding of the bioformation of
myrobotinol (1) in the plant, a plausible biosynthetic pathway
was proposed.
Experimental Section
Plant Material and Extraction. M. nutans Drake was collected
in North Vietnam in June 2000 and a specimen (VN 700) was
deposited in the Institute of Botany-VAST-Vietnam. The dried and
ground leaves (5 kg) were alkalinized with aqueous NH4OH (10%)
and extracted with CH2Cl2. After evaporation to dryness, the CH2-
Cl2 crude extract was suspended in aqueous 5% HCl, then extracted
with CH2Cl2. The crude alkaloid extract (29.5 g) obtained after
solvent removal under reduced pressure was purified by repeated
open column chromatography, eluted with a gradient of CH2Cl2/
MeOH to afford myrobotinol (1, 20 mg).
9828 J. Org. Chem., Vol. 72, No. 25, 2007

Myrobotinol (1): solid, mp 225-226 °C (n-hexane/Et2O); [R]20
(S)-MTPA-Myrobotinol Ester (3). According to the above
procedure, 3 (2.2 mg, 33% yield) was obtained from myrobotinol
D
+36.4 (c 0.4, MeOH); IR (KBr) νmax (cm-1) 3386, 2932, 2866,
1652, 1447, 1407, 1380, 1137, 1117, 1099, 1038, 1016, 993, 955,
921, 852, 827, 721, 599; ESI-MS (TOF), m/z 333.2538 [M + H]+
(calcd.333.2542 for C20H33N2O2); ESI-MSMS (TOF), on [M + H]+
ion, m/z 333, 290, 198, 196, 178, 168, 166, 150, 148, 124, 105, 91.
(R)-MTPA-Myrobotinol Ester (2). (R)-MTPA (10 mg, 0.43
mmol) was treated with SOCl2 and a small amount of NaCl at rt
for 48 h. Excess SOCl2 was removed under diminished pressure,
then the crude was co-evaporated with toluene to complete removal
of SOCl2. The remaining crude was dissolved in dry CHCl3 (0.3
mL) and treated with myrobotinol (1, 3 mg, 0.009 mmol) in 0.1
mL of dry CHCl3, a drop of pyridine was added, and the solution
was stirred for 24 h. The volatile layer was removed under
diminished pressure and the crude was purified by preparative TLC
(CH2Cl2/MeOH 95/5) to afford 2 (3.5 mg, 75%). Colorless oil,
[R]20D +36.4 (c 0.3, MeOH); ESI-MS (TOF), m/z 549.2958 [M +
H]+ (calcd. 549.2942 for C30H40F3N2O4); ESI-MSMS (TOF), on
[M + H]+ ion, m/z 549 [M + H]+, 506, 412, 382, 315, 272, 178,
150, 124; 1H (400.13 MHz, CDCl3, 298 K) 7.49 (m, 2H, H-5′ and
H-9′), 7.38, (m, 1H, H-7′), 7.37 (m, 2H, H-6′ and H-8′), 5.13 (m,
1H, H-16), 4.20 (dd, 9.6 and 2.4 Hz, 1H, H-21), 4.12 (d, 3.3 Hz,
1H, H-11), 4.01 (dd, 10.8 and 4.5 Hz, 1H, H-13eq), 3.5 (s, 3H,
OMe-10′), 3.26 (dd, 10.8 and 10.8 Hz, 1H, H-13ax), 3.11 (dd, 11.8
and 4.7 Hz, 1H, H-10), 2.74 (m, 1H, H-2eq), 2.72 (dd, 11.4 and 4.6
Hz, 1H, H-23), 2.64 (m, 1H, H-2ax), 2.28 (m, 1H, H-14), 2.23 (m,
1H, H-9), 1.99 (m, 1H, H-18), 1.93 (m, 1H, H-5), 1.91 (m, 1H,
H-17eq), 1.84 (m, 1H, H-15eq), 1.73 (m, 1H, H-3eq), 1.69 (m, 2H,
CH2-20), 1.59 (m, 1H, H-17ax), 1.57 (m, 2H, CH2-19), 1.53 (m,
1H, H-4ax), 1.50 (m, 3H, H-3ax and CH2-6), 1.42 (m, 1H, H-8eq),
1.38 (m, 2H, CH2-7), 1.33 (m, 1H, H-8ax), 1.27 (m,1H, H-4eq), 1.01
(ddd, 12.0, 10.6 and 10.5 Hz, 1H, H-15ax); 13C (75.47 MHz, CDCl3,
298 K) 165.8 (C-1′), 132.3 (C-4′), 129.6 (C-7′), 128.4 (C-6′ and
C-8′), 127.3 (C-5′ and C-9′), 91.9 (C-11), 84.9 (C-2′), 72.8 (C-
13), 71.5 (C-16), 68.8 (C-21), 64.2 (C-23), 57.4 (C-10), 55.3 (OMe-
10′), 38.2 (C-2), 36.0 (C-18), 35.6 (C-17), 35.1 (C-5), 32.9 (C-15),
31.4 (C-6), 30.7 (C-9), 30.1 (C-20), 27.5 (C-8), 26.6 (C-14), 26.2
(C-3), 25.2 (C-19), 24.2 (C-4), 20.2 (C-7).
(4 mg) and (S)-MTPA (10 mg). Colorless oil, [R]20 -5 (c 0.25,
D
MeOH); ESI-MS (TOF), m/z 549.2963 [M + H]+ (calcd 549.2942
for C30H40F3N2O4); ESI-MSMS (TOF), on [M + H]+ ion, m/z 549
1
[M + H]+, 506, 412, 315, 298, 285, 272, 190, 178, 150, 124; H
(400.13 MHz, CDCl3, 298 K) 7.49 (m, 2H, H-5′ and H-9′), 7.38
(m, 1H, H-7′), 7.37 (m, 2H, H-6′ and H-8′), 5.13 (m, 1H, H-16),
4.21 (m, 1H, H-21), 4.11 (d, 3.1 Hz, 1H, H-11), 3.99 (dd, 10.9 and
4.5 Hz, 1H, H-13eq), 3.51 (s, 3H, OMe-10′), 3.23 (dd, 10.9 and
10.9 Hz, 1H, H-13ax), 3.11 (m, 1H, H-10), 2.77 (m, 1H, H-2eq),
2.72 (m, 1H, H-23), 2.67, (m, 1H, H-2ax), 2.26, (m, 1H, H-14),
2.22, (m, 1H, H-9), 1.99 (m, 2H, H-18 and H-17eq), 1.93 (m, 1H,
H-5), 1.75 (m, 1H, H-15eq), 1.73 (m, 1H, H-3eq), 1.69 (m, 3H, 17ax
and CH2-20), 1.68 (m, 1H, H-19ax), 1.58 (m, 1H, H-19eq), 1.53 (m,
1H, H-4ax), 1.50 (m, 3H, H-3ax and CH2-6), 1.42 (m, 1H, H-8eq),
1.38 (m, 2H, CH2-7), 1.33 (m, 1H, H-8ax), 1.27 (m, 1H, H-4eq),
0.90 (ddd, 12.0, 11.8 and 8.8 Hz, 1H, H-15ax); 13C (75.47 MHz,
CDCl3, 298 K) 165.8 (C-1′), 132.3 (C-4′), 129.6 (C-7′), 128.4 (C-
6′ and C-8′), 127.2 (C-5′ and C-9′), 91.8 (C-11), 84.5 (C-2′), 72.3
(C-13), 71.5 (C-16), 68.8 (C-21), 64.5 (C-23), 57.4 (C-10), 55.3
(OMe-10′), 38.1 (C-2), 36.0 (C-18), 35.9 (C-17), 35.1 (C-5), 32.6
(C-15), 31.3 (C-6), 30.7 (C-9), 30.0 (C-20), 27.5 (C-8), 26.5 (C-
14), 26.1 (C-3), 25.2 (C-19), 24.2 (C-4), 20.1 (C-7).
Acknowledgment. The authors thank Mr. A. Gramain and
Mr. D. C. Dao (Institute of Chemistry-VAST, Vietnam) for
plant collection and its botanical determination, Dr. A. Blond
and Mr. Lionel Dubost (MNHN, France) for recording 2D NMR
spectra and ESI-MS measurement, respectively, and Prof. P.
Grellier (MNHN, France) for antiplasmodial assay. The CNRS
is gratefully acknowledged for doctoral fellowship support
(V.C.P.).
Supporting Information Available: NMR and MS spectra of
myrobotinol (1). This material is available free of charge via the
Internet at http://pubs.acs.org.
JO7017203
J. Org. Chem, Vol. 72, No. 25, 2007 9829