A formylating agent by dehydration of the natural product DIMBOA

Article abstract of DOI:10.1021/np980515s

The natural aglucone 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA, 1) of maize underwent spontaneous dehydration and rearrangement to form 3-formyl-6-methoxybenzoxazolin-2(3H)-one (FMBOA, 2) on reaction with N- ethoxycarbonyl-trichloroacetaldimine. Compound 2 was proven to be a reactive formyl donor toward N-, O-, and S-nucleophiles, which may be important in case 2 is formed under biological conditions.

Full text of DOI:10.1021/np980515s

J . Nat. Prod. 1999, 62, 1151-1153
1151
A F or m yla tin g Agen t by Deh yd r a tion of th e Na tu r a l P r od u ct DIMBOA
Angelika Hofmann and Dieter Sicker*
Institute of Organic Chemistry, University of Leipzig, Talstrasse 35, D-04103 Leipzig, Germany
Received November 18, 1998
The natural aglucone 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA, 1) of maize
underwent spontaneous dehydration and rearrangement to form 3-formyl-6-methoxybenzoxazolin-2(3H)-
one (FMBOA, 2) on reaction with N-ethoxycarbonyl-trichloroacetaldimine. Compound 2 was proven to
be a reactive formyl donor toward N-, O-, and S-nucleophiles, which may be important in case 2 is formed
under biological conditions.
1
2
3
Crop plants such as maize, rye, and wheat, exhibit a
Sch em e 1. Syntheses of
3-Formyl-6-methoxybenzoxazolin-2(3H)-one (2)
4
natural preinfectional resistance system toward pathogens
structurally based upon acetal 2-â-D-glucosides of the (2R)-
2
,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one skeleton. After
a pest attack, the bioactive aglucones are released by
â-glucosidase and act against the pest. They have also been
5
found to occur in phytotoxic root exudates as a weapon in
the fight between different plant species. The best in-
vestigated aglucone is 2,4-dihydroxy-7-methoxy-2H-1,4-
6
benzoxazin-3(4H)-one (DIMBOA, 1) of maize. Synthetic
7
approaches to 1 have been reviewed, and recently a novel
8
DIMBOA synthesis has been reported. We now report on
the dehydration of 1 with formation of 3-formyl-6-meth-
oxybenzoxazolin-2(3H)-one (FMBOA, 2), which is proven
to be a very reactive formyl donor toward typical nucleo-
philes that occur in biomolecules.
DIMBOA is an enzyme inhibitor of, for example, R-chy-
9
10
motrypsin, aphid cholinesterases, and plasma membrane
an exceptional behavior due to a cascade of spontaneous
reactions. Instead of an adduct, the hitherto unknown
FMBOA (2) was isolated as the product of a formal
dehydration and rearrangement (Scheme 1). (2,2,2-Trichloro-
1-hydroxy-ethyl)-carbamic acid ethyl ester was unequivo-
cally identified as the second product by comparison of the
+
11
H -ATPase. Several proposals for the molecular mode of
action are based on model experiments with 1 and struc-
tural analogues. Hence, it was suggested that the inhibition
may result from reaction with nucleophilic groups such as
-
NH
acetal 1 undergoes an oxo-cyclo tautomerization. The
aldehyde group of the oxo form reacts with the ꢀ-NH group
2
from a lysine or -SH from a cysteine unit. Hemi-
1
mp, H NMR, and IR spectra of a sample isolated from the
1
2
reaction mixture with those of an authentic sample. A H
of N-R-acetyl lysine.12 The importance of subunits of
DIMBOA was studied by removing either the 2-OH group
or the 7-MeO group, together with an activation of the
analogues, by acetylation at N-OH.13 Both model com-
pounds, 4-acetoxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one
and 4-acetoxy-2-hydroxy-2H-1,4-benzoxazin-3(4H)-one, were
shown to react with various C-, N-, or S-nucleophiles,
regioselectively. Even DNA reacted with the hydroxamic
acid N atom.14 Thus, 1 was regarded as a precursor of a
reactive, multi-centered cationic electrophile, generated by
N-O bond heterolysis, which is facilitated by the 7-MeO
group. Hence, the high activity of 1 results from the
combination of both the hemiacetal- and the 7-donor-
activated cyclic hydroxamic acid moieties.
NMR spectrum of the crude reaction product showed it to
consist of equivalent amounts of 2 and (2,2,2-trichloro-1-
hydroxy-ethyl)-carbamic acid ethyl ester.
After initial addition to the imine the combined action
of the strong acceptor unit at N-O together with the donor
effect of the MeO group causes a spontaneous heterolysis
of the N-O bond in the oxo form. As result of a rearrange-
ment sequence, 2 is formed.17 The structure of 2 was
1
unequivocally assigned by means of spectroscopic data. H
NMR showed deshielding for H-4 and a singlet at δ 9.20
ppm, which did not undergo an H-D exchange with D O.
2
This signal corresponds to a carbon signal at 158.4 ppm
with J C,H ) 218.6 Hz both typically for CHO in formamides.
+
The mass spectrum showed a fragment for [M - 16] at
We have now found that another very reactive species
can be obtained from 1 by formal dehydration. Usually,
cyclic hydroxamic acids, including the natural aglucone 2,4-
dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA), an ana-
logue of 1, form stable adducts with azavinylogous N-acyl-
trichloroacetaldimines.15 However, on addition of an
equimolar amount of N-ethoxycarbonyl-trichloroacetaldi-
mine16 to a solution of natural 1 in THF at 20 °C, 1 showed
m/z 177 besides the base peak m/z 165. An NOE between
CHO and H-4 was not observed. This can be explained
assuming a partial double bond between C and N in the
N-formyl unit as is typical for formamides with an orienta-
tion of the O atom toward H-4, thus preventing an NOE of
CHO. Finally, this conformational feature was exactly
1
revealed by X-ray analysis, which showed four molecules
of 2 per unit cell, connected through hydrogen bonds
(Figure 1, Tables 1 and 2).
FMBOA (2) resembles N-formylbenzotriazole, reported
as a reactive formyl donor.18 We have studied the behavior
*
To whom correspondence should be adressed. Tel.: 0049-341-9736574.
Fax: 0049-341-9736599. E-mail: sicker@organik.orgchem.uni-leipzig.de.
1
0.1021/np980515s CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/31/1999

1
152 J ournal of Natural Products, 1999, Vol. 62, No. 8
Notes
F igu r e 1. An ORTEP diagram of 3-formyl-6-methoxybenzoxazolin-
(3H)-one (2).
2
F igu r e 2. CHO part of the 1H NMR in THF-d
of 2 (left), and its
8
reactions with N,N-diethylamine (middle left), MeOH (middle right),
and methyl thioglycolate (right).
Ta ble 1. Crystal Data for 2
empirical formula
C9H7NO4
formula weight
crystal system, space group
unit cell dimensions
193.16
triclinic, P1h
a ) 9.357(1) Å R ) 107.63(1)°
b ) 10.522(1) Å â ) 113.66(1)°
c ) 10.567(1) Å γ ) 99.76(1)°
method.20 In NMR tubes 0.1 mmol of N,N-diethylamine,
MeOH, and methyl thioglycolate dissolved in THF-d have
8
been reacted each with 0.1 mmol of 2 at room temperature.
Spectra measured after 24 h showed almost complete
reactions. The progress of these reactions could be observed
using the signal of the CHO proton, which occurs in THF-
number of formulas per unit cell 4
calculated density
1.499 g/cm3
0.710 73
0.955
R1 ) 0.0509
R1 ) 0.0992
wavelength
8
d at δ 9.12 for 2 and undergoes a distinct shift in the
goodness-of-fit on F 2
products formed, as in N,N-diethylformamide (δ 7.97),
methyl formiate (δ 8.02), and methyl S-methoxycarbonyl
thioformiate (δ 10.15) (Figure 2). A preparative formylation
of benzylamine is described in the Experimental Section.
Therefore, 2 is a reactive formyl donor toward N-, O- and
S-nucleophiles comparable to N-formylbenzotriazole.
final R indices [I > 2σ(I)]
R indices (all data)
wR2 ) 0.1125
wR2 ) 0.1350
4
Ta ble 2. Atomic Coordinates (× 10 ) and Equivalent Isotropic
2
3
Displacement Parameters (A × 10 ) for 2
x
y
z
U(eq)a
The dehydration sequence of 1 discussed offers yet
another interpretation of the bioactivity mode of 1. Under
natural conditions 1, after metabolic N-OH acylation
followed by N-O heterolysis may, if a nucleophile is not
immediately present, rearrange to 2 as a potent formylat-
O(1)
O(2)
O(3)
O(4)
O(5)
O(6)
O(7)
O(8)
N(1)
N(2)
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(8)
C(9)
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
C(17)
C(18)
3919(2)
9980(2)
3567(3)
1317(2)
2244(2)
2436(2)
7817(3)
3277(2)
3625(3)
5003(3)
6750(3)
8266(4)
8400(3)
6991(3)
5493(3)
5353(3)
2767(3)
10 171(5)
2832(4)
5609(4)
4793(3)
3082(3)
2091(3)
2950(3)
4632(3)
3508(3)
681(4)
8058(2)
9364(2)
3840(2)
6686(2)
1456(2)
-3199(2)
3104(2)
3738(2)
5949(2)
2366(2)
6219(3)
7186(3)
8511(3)
8903(3)
7905(3)
6615(3)
6861(3)
10 764(3)
4636(3)
72(3)
8486(2)
11 419(2)
5299(2)
6580(2)
6475(2)
3808(2)
9604(2)
8215(2)
6834(2)
8071(2)
8106(3)
9320(3)
10 301(3)
10 116(3)
8909(3)
7925(3)
7198(3)
12 369(4)
5591(3)
6975(3)
5847(3)
4872(3)
4984(3)
6128(3)
7091(3)
7673(3)
2788(4)
9245(3)
40(1)
46(1)
51(1)
50(1)
41(1)
42(1)
56(1)
46(1)
34(1)
33(1)
37(1)
39(1)
34(1)
36(1)
33(1)
32(1)
38(1)
51(1)
44(1)
34(1)
33(1)
33(1)
33(1)
32(1)
31(1)
39(1)
52(1)
46(1)
ing agent toward -NH , -OH, or -SH groups. Therefore,
2
some of the biological effects observed could also result from
formylations of biomolecules.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
measured on a Boetius micro hot-stage and are corrected. The
1
NMR spectra were recorded with a Varian Gemini 200 ( H,
199.975 MHz; 13C, 50.289 MHz) spectrometer with hexameth-
yldisiloxane as the internal standard. The MS was recorded
on a VG Masslab Manchester VG 12-250 spectrometer (70
eV EI ionization). The elemental analysis was performed on a
Heraeus CHN-O-Rapid analyzer. For the X-ray structure
determination a SIEMENS CCD diffractometer with a graph-
ite monochromator and Mo KR radiation was used. The
intensity data were collected at 223 K; 4524 reflections (were)
collected with 2 < θ < 27°, of these 3199 were independent
(Rint ) 0.032). All 1946 observed reflections [I(hkl) > 2 σ(I)]
were used for determination of the unit cell parameters. The
structures were solved by direct methods (SHELX-97) and
subsequent difference Fourier synthesis and refined by full-
matrix least-squares on F2 (SHELX-97). The H atoms were
located by difference Fourier map and refined isotropically.
The starting DIMBOA (1) was isolated from frozen maize
-1300(3)
-1812(3)
-955(3)
405(3)
930(3)
2668(3)
-3755(4)
3369(3)
6527(4)
a
U(eq) is defined as one-third of the trace of the orthogonalized
Uij tensor.
of 3 toward model nucleophiles. To obtain 2 in larger
quantities, an independent synthesis, starting from 6-meth-
oxy-benzoxazolin-2(3H)-one (MBOA, 3),19 was accomplished
with in-situ-prepared acetylformylanhydride in THF solu-
tion in the presence of 4-DMAP, adapting a literature
6
seedlings according to the procedure reported. N-Ethoxycar-
bonyl-trichloroacetaldimine was prepared following the litera-
ture method.16 MBOA (3) was accessible from 5-methoxy-2-
nitrophenol in two steps.19

Notes
J ournal of Natural Products, 1999, Vol. 62, No. 8 1153
3
-F or m yl-6-m eth oxyben zoxa zolin -2(3H)-on e (2) fr om
55 °C (lit.21 mp56-58 °C), its NMR identical with that of an
authentic sample.
DIMBOA (1). To a stirred solution of DIMBOA (1) (0.5 mmol,
1
06 mg) in dry THF (15 mL) was added a solution of
N-ethoxycarbonyl-trichloroacetaldimine (0.5 mmol, 109 mg) in
dry THF (5 mL) in one portion at 20 °C. After 1 h the solvent
was removed in vacuo and the oily residue triturated with
EtOH (5 mL) to cause crystallization. The crude pale-brown
Ack n ow led gm en t. We thank Ms. S. J elonek for the X-ray
structure determination and Dr. L. Hennig for NMR measure-
ments. The financial support for this work by the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen In-
dustrie is gratefully acknowledged.
crystals were recrystallized from CHCl
3
to yield 3-formyl-6-
methoxybenzoxazolin-2(3H)-one (3) (43 mg, 45%) as off-white
1
crystals: mp 144-146 °C; H NMR (200 MHz, CDCl
3
) δ 9.20
Su p p or t in g In for m a t ion Ava ila b le: A scheme showing pro-
posed mechanism of formation of 2 by dehydration of 1, tables of
crystal structure information of 2, and a figure showing the unit
cell of 2. This material is available free of charge via the Internet
at http://pubs.acs.org.
(
s, 1H, CHO), 7.82 (d, 1H, H-4), 6.83 (d, 1H, H-7), 6.78 (dd,
1
3
1
H, J 4,5 ) 9.0 Hz, J 5,7 ) 2.2 Hz, H-5), 3.83 (s, 3H, OCH
3
);
C
NMR (50 MHz, CDCl
3
) δ 158.7 (C-6), 158.4 (CHO), 152.7 (C-
2
7
1
), 143.9 (C-7a), 119.6 (C-4a), 115.4 (C-4), 110.7 (C-5), 98.1 (C-
+
), 56.4 (OCH
50 (70), 136 (12); anal. C 56.30%, H 3.97%, N 7.56%, calcd
NO , C 55.96%, H 3.65, N 7.25%.
3
), EIMS, m/z 193 [M] (23), 177 (11), 165 (100),
Refer en ces a n d Notes
for C
9
H
7
4
(1) Hartenstein, H.; Klein, J .; Sicker, D. Ind. J . Heterocycl. Chem. 1993,
For identification, (2,2,2-trichloro-1-hydroxy-ethyl)-carbamic
acid ethyl ester was isolated by column chromatography (Si
2, 151-153.
(
(
2) Hartenstein, H.; Sicker, D. Phytochemistry 1994, 35, 827-828.
3) Kluge, M.; Grambow, H. J .; Sicker, D. Phytochemistry 1997, 44, 639-
gel 60 0.040-0.063 mm, Merck, eluent isohexane-CHCl
3
1:1)
6
41.
1
6
from the remaining residue: mp 99-100 °C (lit. mp 103 °C),
(
4) Niemeyer, H. M. Phytochemistry 1988, 27, 3349-3358.
1
H NMR (200 MHz, CDCl
q, 2H, J ) 7.0 Hz, -OCH
identical with the spectrum of an authentic sample).
-F or m yl-6-m eth oxyben zoxa zolin -2(3H)-on e (2) fr om
MBOA (3). At 0 °C to Ac O (25 mmol, 1 mL) 100% HCO
12.5 mmol, 0.5 mL) was added. The mixture was warmed to
3
) δ 5.68 (s, 1H, Cl
3
C-CH-), 4.20
(5) Friebe, A.; Schulz, M.; K u¨ ck, P.; Schnabl, H. Phytochemistry 1995,
8, 1157-1159.
6) Hartenstein, H.; Klein, J .; Sicker, D. Ind. J . Heterocycl. Chem. 1993,
, 151-153.
3
(
(
2
-), 1.28 (t, 3H, J ) 7.0 Hz, -CH
3
),
(
2
3
(7) Sicker, D.; Hartenstein, H.; Kluge, M. Natural Benzoxazinoids-
Synthesis of Acetal Glucosides, Aglucones, and Analogues. In Recent
Research Developments in Phytochemistry; Pandalai, S. G., Ed.;
Research Signpost: Trivandrum, India, 1997; pp 203-223.
2
2
H
(
5
0 °C, kept for 15 min at this temperature, and cooled in an
(
8) Tays, K.; Atkinson, J . Synth. Commun. 1998, 28, 903-912.
ice bath to 0 °C again. A solution of 4-(dimethylamino)pyridine
1 mmol, 122 mg) in dry THF (30 mL) was added. To the
rapidly stirred solution, 6-methoxybenzoxazolin-2(3H)-one (3)
10 mmol, 1.65 g) was added and stirred for 1 h at 0 °C and 14
(9) Cuevas, L.; Niemeyer, H. M.; Perez, F. J . Phytochemistry 1990, 29,
1429-1432.
(
(
(
10) Cuevas, L.; Niemeyer, H. M. Phytochemistry 1993, 34, 983-985.
11) Friebe, A.; Roth, U.; K u¨ ck, P.; Schnabl, H.; Schulz, M. Phytochemistry
(
1
997, 44, 979-983.
h at 20 °C. The solvent was removed in vacuo, and the solid
residue was stirred with EtOH (12 mL) and filtrated. Off-white
crystals of 3-formyl-6-methoxybenzoxazolin-2(3H)-one (3) (1.14
g, 59%) remained, identical in their properties with 2 prepared
from 1.
(12) Perez, F. J .; Niemeyer, H. M. Phytochemistry 1989, 28, 1831-1834.
(
(
13) Hashimoto, Y.; Shudo, K. Phytochemistry 1996, 43, 551-559.
14) Hashimoto, Y.; Ishizaki, T., Shudo, K. Tetrahedron 1991, 47, 1837-
1
860.
(
15) Sicker, D.; Hartenstein, H. Z. Chem. 1989, 29, 169-170.
(16) Ulrich, H.; Tucker, B.; Sayigh, A. A. R. J . Org. Chem. 1968, 33, 2887-
2
889.
F or m yla tion of Ben zyla m in e w ith 2. To a solution of 2
(
(
17) See Supporting Information for a mechanistic proposal.
(386 mg, 2 mmol) in dry THF (30 mL) benzylamine (215 mg,
18) Katritzky, A. R.; Chang, H.-X.; Yang, B. Synthesis 1995, 503-505.
2
mmol) was added. The solution was refluxed for 30 min. The
(19) Sicker, D. Synthesis 1989, 875-876.
solvent was removed in vacuo, and from the solid residue
N-benzyl-formamide was extracted with refluxing n-hexane
(20) Tavernier, D.; van Damme, S.; Ricquier, P.; Anteunis, M. J . O. Bull.
Soc. Chim. Belg. 1988, 97, 859-865.
(
21) Cooks, R. G.; Sykes, P. J . Chem. Soc. C 1968, 2864-2871.
(
7 × 25 mL). On cooling to 0 °C the N-benzyl-formamide
crystallized to form off-white needles (184 mg, 68%), mp 53-
NP980515S