Isolation and synthesis of an α-malamic acid derivative from Justicia ghiesbreghtiana

Article abstract of DOI:10.1021/np9801662

A polar extract of leaves of Justicia ghiesbreghtiana yielded N-(2- hydroxy-4,5-dimethoxyphenyl)(S)-α-malamic acid, 1. Incomplete spectral analysis yielded a hypothetical structure, which was then proven by total synthesis. Coupling of the trifluoroacetate of malic anhydride (trifluoroacetoxysuccinic anhydride) with an arylamine provided the key to regiospecific preparation of the α- rather than β-malamic acids.

Full text of DOI:10.1021/np9801662

1174
J . Nat. Prod. 1998, 61, 1174-1176
Isola tion a n d Syn th esis of a n r-Ma la m ic Acid Der iva tive fr om
J u sticia gh iesbr egh tia n a
Lotfy D. Ismail,† Peter Lorenz,‡ and Frank R. Stermitz*,‡
Department of Pharmacognosy, Al-Azhar University, Nasr City, Cairo, Egypt, and Department of Chemistry,
Colorado State University, Fort Collins, Colorado 80523-1872
Received April 27, 1998
A polar extract of leaves of J usticia ghiesbreghtiana yielded N-(2-hydroxy-4,5-dimethoxyphenyl)-
(S)-R-malamic acid, 1. Incomplete spectral analysis yielded a hypothetical structure, which
was then proven by total synthesis. Coupling of the trifluoroacetate of malic anhydride
(trifluoroacetoxysuccinic anhydride) with an arylamine provided the key to regiospecific
preparation of the R- rather than â-malamic acids.
Several specimens of an Acanthaceous plant labeled
J usticia ghiesbreghtiana Lem., of presumed Mexican
origin, are found worldwide in botanical or horticultural
gardens, but the connection between these specimens
and collection data is obscure. The name J usticia
ghiesbregtiana was originally published in 18471 by
Lemaire, who stated that the specimen he saw was
“introduced from Mexico by the naturalist M. Ghies-
bregt”. The collector was, more properly, August Ghies-
breght, and the name has therefore been preserved as
J . ghiesbreghtiana. It was not mentioned in the most
recent comprehensive treatment of J usticia, although
not all of the some 600 species of J usticia are discussed
therein.2 J . ghiesbreghtiana is sometimes treated (par-
ticularly in the botanical garden literature) as synony-
mous with the better known Mexican species J . spici-
gera Schlechtend., which has been used as a medicinal
herb in Mexico3 and cultivated as such in Texas.4 J .
spicigera is said to be used as a stimulant, to treat
scabies and various gastrointestinal disorders such as
dysentery, and as a source of blue dye.2-5 Chemical
investigations of J . spicigera reported the presence of
the flavanoid kaempferitrin,4 â-sitosterol glucoside,
cryptoxanthin, alantoin, and a black, resinous dye.3 We
report here isolation of a novel malamic acid derivative
from a Cairo, Egypt, botanical garden specimen of J .
ghiesbreghtiana.
A water-soluble fraction remaining after removal of
nonpolar materials and extensive column chromatog-
raphy yielded a pure (TLC, NMR), gummy compound,
C12H15NO7 by HRMS. The 13C and 1H NMR spectra
accounted for the presence of two carbonyls, two meth-
oxy groups on a tetrasubstituted benzene, one methyl-
ene, and one methine to which an oxygen was attached.
Two 1H singlets at δ 6.50 and 6.94 suggested that the
benzene ring was 1,2,4,5-substituted. A base peak of
m/ z 154 in the HRMS was due to a C7H8NO3 fragment,
while a peak of second intensity at m/ z 169 was
established as C8H11NO3. These formulas suggested
that the aromatic portion of the unknown was substi-
tuted with two methoxy, one hydroxy, and one -NHR
group. The 13C NMR resonances seemed best to fit a
2-hydroxy-4,5-dimethoxyaniline derivative, although a
2,5-dimethoxy-4-hydroxyaniline could not be absolutely
ruled out. Cleavage at the N-R bond in the mass
spectral fragmentation with concomitant rearrangment
of one H from the side chain would yield the m/z 169
1
ion. Decoupling of the H NMR spectrum, along with
the chemical shifts of the methine and methylene
protons, suggested the presence of a -CO-CH(OH)-
CH2-CO- moiety attached at one carbonyl to the
aniline NH. The IR spectrum showed a complex series
of absorptions in the 1550-1650 cm-1 region, suggesting
amide, carboxyl, and/or carboxylate functionalities. The
data to this point were interpreted as best being
consistent with either structure 1 or 2 for the unknown,
where the absolute stereochemistry at the side chain
OH was that expected if it were derived from L-malic
acid. Compounds 1 and 2 are R- and â-malamic acids,
respectively.
During the course of obtaining the spectral data and
allowing the unknown to stand in solution, the aromatic
proton resonances eventually disappeared and were
replaced by singlet resonances at δ 5.45 and 5.55, with
the remainder of the spectrum not greatly changed. MS
of the product showed the loss of two hydrogens from
the original formulation and suggested transformation
of the unknown into an acyl quinone-imine structure
3.6 This transformation led to decomposition, and more
detailed spectral studies were not possible.
At this point, several R- and â-malamic acids analo-
gous to structures 1 and 2 were synthesized in an
attempt to discover unique spectral data for each that
would allow assignment of either regiochemistry to the
* To whom correspondence should be addressed. Tel.: (970) 491-
5158. Fax: (970) 491-5610. E-mail: frslab@lamar.colostate.edu.
† Al-Azhar University.
‡ Colorado State University.
S0163-3864(98)00166-9 CCC: $15.00
© 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/31/1998

Notes
J ournal of Natural Products, 1998, Vol. 61, No. 9 1175
Sch em e 1
CDCl3 unless otherwise indicated. IR spectra were from
thin films on NaCl unless otherwise indicated. Organic
extraction solvents were dried over Na2SO4 and evapo-
rated in vacuo.
P la n t Ma ter ia l. Fresh plant material of J usticia
ghiesbreghtiana Lem. was collected on December 16,
1992, in the Giza zoological garden, Cairo, Egypt. It
was identified by Professor Dr. Nabeil El Hadedi,
Department of Botany, Cairo University, and a voucher
was deposited in the Department of Pharmacognosy
herbarium, Al-Azhar University.
Extr a ction a n d Isola tion . Air-dried and powdered
aerial parts (700 g) were extracted with 3:2 hexane-
EtOAc and the marc dried and extracted with MeOH.
The MeOH was evaporated, the residue was triturated
with H2O, and the aqueous layer was washed with Et2O
and evaporated to dryness. The residue was triturated
with MeOH, and the MeOH was then evaporated to a
gummy residue (29 g). This was dissolved in H2O,
chromatographed on a C18 Si gel column, and eluted
with H2O-MeOH. Fractions 4-8 (5-25% MeOH)
showed orange-red spots on TLC with 2,4-DNPH re-
agent. These were combined and evaporated to leave
2.1 g of residue. This was chromatographed on Si gel
with CHCl3-MeOH as eluting solvent. Fractions 21-
33 (45-70% MeOH) were combined to yield 0.85 g of
residue, which was similarly rechromatographed. Frac-
tion 9 (25% MeOH; 130 mg) gave a single spot on Si gel
TLC (Rf 0.47; solvent CHCl3-MeOH-H2O 61:32:7) as
an almost colorless amorphous solid, eventually identi-
unknown. The results were not unequivocal and will
be reported elsewhere, but they suggested that the
R-malamide regiochemistry was the proper one for the
unknown. A total synthesis of 1 was therefore under-
taken (Scheme 1). The key to the regiospecific synthesis
was coupling of the amine 5 with (S)-trifluoroacetoxy-
succinic anhydride.7,8 The final product acid had NMR
spectra very similar to, but not identical with, those of
the originally isolated unknown. The NMR spectral
data and optical rotation became essentially identical
when the synthetic product was converted to the am-
monium salt, thus indicating that the original isolation
had resulted in isolation of 1 as a salt. Both the isolated
and synthetic 1 were originally obtained as almost
colorless or with a faint pinkish or purplish cast, but
upon obtaining spectra, or even storage in the refrigera-
tor, they became a darker blue-purple.
J usticia species, particularly some used as medicinal
herbs, often contain lignans, but we were unable to
isolate any from J . ghiesbreghtiana. One medicinal
species, J . gendarussa, was also reported to be lignan-
free, but did contain two 2-aminobenzyl alcohol deriva-
tives,9 the only other aminobenzene derivative we could
find reported from J usticia. Structurally, and perhaps
biosynthetically, 1 may be related to the benzoxazoli-
nones and 1,4-benzoxazinones common to seedlings of
the Gramineae. One such (2,4-dihydroxy-1,4-benzox-
azin-3-one) was reported from Acanthus mollis of the
same family as J usticia (Acanthaceae).10
fied as a salt of 1: [R]20 -23° (c 1.7, H2O); IR (KBr)
D
ν
max 3445 (br), 1640, 1610, 1535 cm-1; 1H NMR (D2O) δ
2.56 (dd, 1H, J ) 8.0, 15.5 Hz), 2.70 (dd, 1H, J ) 4.0,
15 Hz), 3.67 (s, 3H), 3.71 (s, 3H), 4.51 (dd, 1H, J ) 4.0,
8.0 Hz), 6.50 (s, 1H), 6.94 (s, 1H); 13C NMR (D2O) δ 41.9,
56.8, 57.3, 70.1, 102.2, 110.2, 116.0, 142.4, 144.4, 148.2,
175.9, 178.6; negative EIMS (electrospray) m/z 284
(100); positive EIMS (electrospray) m/z 286 (30), 214
(100); HRFABMS m/z 285.0844 (calcd for C12H15NO7
285.0849); HREIMS m/z 169.0739 (calcd for C8H11NO3
169.0739) and m/z 154.0506 (calcd for C7H9NO3
154.0504).
Isolated 1 became colored (eventually blue-purple)
upon standing and/or as spectral data were obtained.
It eventually converted completely to a substance
1
(presumably 3) whose aromatic H resonances (δ 6.50
and 6.94) were replaced by two singlets at δ 5.45 and
5.55. This material underwent further decomposition.
2-ter t-Bu t yld im et h ylsilyloxy-3,4-d im et h oxyn i-
tr oben zen e (4). A solution of 3,4-dimethoxy-6-nitro-
phenol11 (3.0 g, 15 mmol), tert-butyldimethylsilyl chlo-
ride (2.41 g, 16.0 mmol) and pyridine (50 mL) was
stirred for 24 h. The pyridine was removed in vacuo
and the crude oily residue purified by VLC (vacuum
layer chromatography) on Si gel (n-hexane-EtOAc,
EtOAc gradient) to give 3.95 g (84%) of 4 as a yellow
solid (purity by GC, 98%): mp 63-65 °C; IR (νmax) 2932,
2859, 1620, 1583, 1514, 1336, 1273, 1222, 1086, 1006,
838 cm-1; 1H NMR δ 0.22 [s, 6H, Si(CH3)2], 1.02 [s, 9H,
SiC(CH3)3], 3.88 (s, 3H, OCH3), 3.89 (s, 3 H, OCH3), 6.41
(s, 1H, CH aromat), 7.48 (s, 1H, CH aromat); 13C NMR
δ -4.5 [Si(CH3)2], 18.2 (C), 25.6 (CH3), 56.2 (OCH3), 56.3
(OCH3), 105.3 (C-3), 107.7 (C-6), 132.8 (C-1), 143.0 (C-
5), 146.1 (C-2), 154.2 (C-4); EIMS m/z 313 [M+] (2), 298
Compound 1 was inactive as an antimicrobial when
tested against Gram-positive and Gram-negative bac-
teria and a fungus. A synthetic analogue lacking the
C-2 OH group was inactive against Mycobacterium
tuberculosis. Almost all solutions of extracts and partial
isolates in this study were dark wine to purple colored,
reflecting the known use of several J usticia species for
the preparation of dyes.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. NMR spectra
were obtained at 300 MHz (1H) and 75 MHz (13C) in

1176 J ournal of Natural Products, 1998, Vol. 61, No. 9
Notes
(24), 258 (38), 257 (65), 256 (100), 240 (38), 211 (57),
196 (26), 183 (26), 166 (16), 137 (6), 75 (28); anal. C
53.65%, H 7.40%, N 4.47%, calcd for C14H23NO5Si C
53.91%, H 7.47%, N 4.53%.
the combined and dried EtOAc extracts, the residue was
purified by VLC as above to give pink crystals. These
were washed with CH2Cl2 to yield 1 (0.35 g, 82%): (mp
175-176 °C); [R]D -56° (c 0.98, MeOH); UV (MeCN)
λmax. (log ꢀ) 302 (7009), 252 (7249); IR (νmax) 3360, 2971,
25
2-ter t-Bu tyld im eth ylsilyloxy-4,5-d im eth oxya m i-
n oben zen e (5). A mixture of 4 (3.5 g, 11 mmol), Pd/C,
10% Pd (0.22 g), and THF (60 mL) was stirred under
H2 for 12 h. After filtration, the solvent was evaporated.
VLC of the residue on Si gel (n-hexane-EtOAc; EtOAc
2939, 1720, 1657, 1525, 1454, 1204, 1001, 858 cm-1; 1H
NMR (DMSO-d6) δ 2.47 (dd, 1H, J ) 8.4, 15.9 Hz, H-9),
2.76 (dd, 1H, J ) 3.9, 15.9 Hz, H-9), 3.65 (s, 3H, OCH3),
3.68 (s, 3H, OCH3), 4.39 (dd, 1H, J ) 3.9, 8.4 Hz, H-8),
6.55 (s, 1H, H-3), 7.85 (s, 1H, H-6), 9.19 (s, 1H, NH);
13C NMR (DMSO-d6) δ 39.6 (CH2), 55.7 (OCH3), 56.3
(OCH3), 68.7 (CH), 101.1 (CH), 105.7 (CH), 118.5 (CNH),
140.4 (COR), 141.1 (COR), 145.2 (COR), 170.8 (CdO),
172.2 (CdO); positive HRCIMS m/z 286.0928 (calcd for
C12H16NO7 286.0926); positive CI m/z 285 [M+] (33), 267
(25), 196 (18), 180 (13), 169 (100), 168 (44), 154 (81),
140 (21), 126 (19), 109 (17), 71 (19), 69 (25); anal. C
50.20%, H 5.55%, N 4.61%, calcd for C12H15NO7, C
50.53%, H 5.30%, N 4.91%.
gradient) gave 5 as an beige oil (2.75 g, 87%): IR (νmax
)
3363, 2930, 2857,1621, 1518, 1465, 1230, 1200, 1013,
1
907, 838, 782 cm-1; H NMR δ 0.27 [s, 6H, Si(CH3)2],
1.03 [s, 9H, SiC(CH3)3], 3.84 (s, 3 H, OCH3), 3.88 (s, 3
H, OCH3), 6.47 (s, 1 H, aromat CH), 7.96 (s, 1 H, aromat
CH); 13C NMR δ -4.4 [Si(CH3)2], 18.0 (C), 25.5 (CH3),
56.1 (OCH3), 56.3 (OCH3), 103.1 (CH), 104.6 (CH), 119.5
(CNH), 138.4 (COR), 143.4 (COR), 146.4 (COR); positive
HRCIMS m/z 284.1695 (calcd for C14H26NO3Si, 284.1682);
GC-MS m/z 283 [M+] 51, 268(22), 226(98), 210(16), 195-
(50), 180(19), 168(14), 73(100).
The acid 1 (31 mg, 0.11 mmol) was dissolved in 1 mL
of concentrated aqueous NH3, evaporated, and dried to
yield 33 mg of the ammonium salt as a pale reddish solid
whose color gradually changed to a deeper, more
N-(2-ter t-Bu t yld im et h ylsilyloxy-4,5-d im et h oxy-
p h en yl)-(S)-r-m a la m ic a cid (6). Trifluoroacetic an-
hydride (TFAA) (2.02 g, 9.62 mmol) was added dropwise
at 0 °C and stirring to (S)-malic acid (0.43 g, 3.2 mmol).
Stirring was continued for 1 h at 0 °C when excess
TFAA was distilled off under reduced pressure. The
obtained anhydride solid residue was dried for 45 min
(oil pump vacuum), dissolved at 0 °C in THF (15 mL),
and a solution of 5 (1.80 g, 6.35 mmol) in THF (15 mL)
was added dropwise under stirring. The reaction mix-
ture was allowed to warm to room temperature and was
stirred for another 1 h. The solvent was removed in
vacuo, and the crude residue was purified by VLC on
Si gel (n-hexane-EtOAc, gradient EtOAc) to yield 6
(0.91 g, 72%): UV (MeCN) λmax (log ꢀ) 304 (6960), 254
(8914); IR (νmax) 3370, 2933, 2859, 1719, 1659, 1534,
purplish color: mp 169-171° dec; [R]25 -26° (c, 0.43,
D
MeOH). Its H and 13C NMR spectra were essentially
1
identical to those of the material originally isolated from
the plant.
Ack n ow led gm en t. This work was supported by
National Science Foundation grants CHE-9321977 and
CHE-9619213. Some mass spectra were obtained on
instruments supported by National Institutes of Health
shared instrumentation grant GM49631. We thank
J ohn J . Beck for preliminary synthetic studies.
Refer en ces a n d Notes
(1) Lemaire, C. Fl. Serres J ard. Eur. 1847, 3, 242.
(2) Graham, V. A. W. Kew Bull. 1988, 43, 551-624. See p 612.
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D′Amore, A. R. Rev. Latinoamer. Qu´ım. 1990, 21, 142-143.
(4) Euler, K. L.; Alam, M. J . Nat. Prod. 1982, 45, 220-221.
(5) Langman, I. K. A Selected Guide to the Literature on the
Flowering Plants of Mexico; University of Pennsylvania: Phila-
delphia, 1964; p 734.
1
1203, 928, 840 cm-1; H NMR δ 0.24 [s, 6H, Si(CH3)2],
1.00 [s, 9H, SiC(CH3)3], 2.77 (dd, 1H, J ) 9.0, 17.4 Hz,
H-9), 3.13 (dd, 1H, J ) 3.2, 17.4 Hz, H-9), 3.81 (s, 3H,
OCH3), 3.83 (s, 3H, H-8), 4.66 (dd, 1H, J ) 3.0, 9.0 Hz,
CH), 6.44 (s, 1H, H-3), 8.00 (s, 1H, H-6), 9.15 (s, 1H,
NH); 13C NMR δ -4.3 [Si(CH3)2], 18.0 (C), 25.6 (CH3),
38.3 (CH2), 56.1 (OCH3), 56.3 (OCH3), 68.6 (CH), 103.6
(CH), 104.8 (CH), 121.1 (CNH), 138.4 (COR), 143.1
(COR), 145.4 (COR), 169.7 (CdO), 176.4 (CdO); positive
HRCIMS m/z 400.1806 (calcd for C18H30NO7Si 400.1792).
N-(2-Hyd r oxy-4,5-d im eth oxyp h en yl)-(S)-r-m a la -
m ic a cid (1). A mixture of 6 (0.60 g, 1.5 mmol) in THF
(10 mL) and 5% HCl (25 mL) was stirred for 12 h. The
THF was removed in vacuo, the aqueous solution was
extracted with EtOAc (3 × 20 mL). After removal of
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NP9801662