Synthesis of 2-[4′-(Ethylcarbamoyl)phenyl]-N-acetylglycine, the proposed structure for giganticine

Article abstract of DOI:10.1021/np010046l

A compound (1) with the structure proposed for giganticine, an antifeedant principle isolated from the root bark of Caloropis gigantea, has been successfully synthesized by two independent methods. Comparison of physical properties and spectroscopic data of 1 with giganticine revealed that they are different compounds. All available evidence suggests that the proposed structure of giganticine is incorrect.

Full text of DOI:10.1021/np010046l

1114
J . Nat. Prod. 2001, 64, 1114-1116
Syn th esis of 2-[4′-(Eth ylca r ba m oyl)p h en yl]-N-a cetylglycin e, th e P r op osed
Str u ctu r e for Giga n ticin e
Chaturong Suparpprom and Tirayut Vilaivan*
Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road,
Patumwan, Bangkok 10330, Thailand
Received J anuary 31, 2001
A compound (1) with the structure proposed for giganticine, an antifeedant principle isolated from the
root bark of Caloropis gigantea, has been successfully synthesized by two independent methods.
Comparison of physical properties and spectroscopic data of 1 with giganticine revealed that they are
different compounds. All available evidence suggests that the proposed structure of giganticine is incorrect.
Sch em e 1
Giganticine is a nitrogen-containing compound isolated
from the root bark of Calotropis gigantea L. collected in
India.1 It exhibited significant antifeedant activity against
Schistocerca gregaria Froskal. The structure of giganticine
was proposed to be 2-[4′-(ethylcarbamoyl)phenyl]-N-acetylg-
lycine (1) based on spectroscopic evidence (IR, MS, 1H
NMR, 13C NMR, and COSY experiments).1 We were
interested in synthesizing this simple amino acid and its
derivatives in order to validate the proposed structure and
also to study structure-antifeedant activity relationships.
However, we have found that synthetic 1 showed very
different physical properties and spectroscopic data from
those of giganticine, details of which are discussed below.
Although the stereochemistry of giganticine was not
established, the L-enantiomer of phenylglycine was arbi-
trarily chosen as a suitable starting material for the
synthesis of giganticine. Direct nitration of phenylglycines
using a concentrated nitric acid-sulfuric acid mixture was
reported to give predominantly the meta-nitrophenylgly-
cine,2 while nitration of N-acetylphenylglycine gave the
para-isomer as the major product.3 L-phenylglycine was
thus converted to N-acetyl-L-(4-aminophenyl)glycine (2) by
an acetylation-nitration-reduction sequence according to
the literature procedure in 28% overall yield. Ethoxycar-
bonylation of 2 with ethyl chloroformate-K2CO3 in aqueous
dioxane followed by acidification gave L-1 as a white
crystalline solid in 35% yield after recrystallization from
ethyl acetate (Scheme 1).
Ta ble 1. Comparison of 1H and 13C NMR Spectra of
Giganticine and L-1
giganticinea,b
L-1c,d
position
δH
δC
δH
δC
1
2
11.32 (1H, s)
1.60 (1H, s)
170.5
91.0
173.0
56.3
5.20 (1H, d,
J ) 7.5 Hz)
1′
2′,6′
128.8
128.2
131.6
128.8
7.50 (2H, d,
J ) 7 Hz)
7.24 (2H, d,
J ) 7 Hz)
7.26 (2H, d,
J ) 8 Hz)
7.46 (2H, d,
J ) 8 Hz)
3′,5′
121.9
118.8
4′
1′′
137.6
60.4
139.7
60.6
4.25 (2H, q,
J ) 7 Hz)
4.10 (2H, q,
J ) 7 Hz)
1.22 (3H, t,
J ) 7 Hz)
8.51 (1H, d,
J ) 7.5 Hz)
9.64 (1H, s)
2′′
1.35 (3H, t,
J ) 7 Hz)
11.24 (1H, s)
14.5
14.8
NH-2
NH-4′
MeCO
EtOCO
COMe
5.57 (1H, brs)
169.9
168.6
27.1
169.9
154.3
22.6
2.36 (3H, s)
1.86 (3H, s)
a
b
In CDCl3 at 300 MHz (1H). Data obtained from ref 1. c In
DMSO-d6 at 200 MHz (1H). Assignment was made based on
d
HMQC, HMBC, and NOESY experiments.
signal of L-1 appeared at δ 5.20, while the H-2 signal of
giganticine appeared at an unusually upfield position (δ
1.60). The C-2 signal L-1 appeared at δ 56.3, which is more
upfield than the corresponding C-2 signal in giganticine
(δ 91.0). The position of 1H and 13C signals at the 2 position
of L-1 are in good agreement with the usual range for
N-acylated R-substituted phenylglycine derivatives.4 Fur-
thermore, the multiplicity of the H-2 signal in L-1 is a
doublet (J ) 7 Hz) due to the spin-spin coupling with the
adjacent NH proton, which is in good agreement with the
structure. In contrast, the same H-2 signal in giganticine
is a singlet and also appeared in an unusual range. In
Synthetic L-1 gave a correct combustion analysis for
C13H16N2O5 (C, H, N) and showed a correct mass at m/z )
280 (EI). However, it exhibited a different IR spectrum
from that of giganticine, especially the absorption due to
CdO stretching (1700 cm-1 for L-1 and 1728 cm-1 for
1
giganticine). The H and 13C NMR spectra of L-1 (DMSO-
d6, 200 MHz) and giganticine (CDCl3, 300 MHz) were also
different, most notably at the position 2 (Table 1). The H-2
* To whom correspondence should be addressed. Tel: +66 2 2184961.
Fax: +66 2 2541309. E-mail: vtirayut@chula.ac.th.
10.1021/np010046l CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/26/2001

Notes
J ournal of Natural Products, 2001, Vol. 64, No. 8 1115
Sch em e 2
were performed on a J EOL J NM-A500 NMR spectrometer.
Chemical shifts are reported in parts per million (ppm, δ) and
are internally referenced to the residual protonated solvent
peak. LREIMS spectra were obtained on a Fisons Instruments
model Trio 2000 mass spectrometer at 70 eV. Elemental
analyses were performed on a Perkin-Elmer elemental ana-
lyzer 2400 CHNS/O at the Research Equipment Centre,
Chulalongkorn University. All chemicals and solvents were
obtained from commercial suppliers (Aldrich, Fluka, and
Merck) and were used as received.
P r ep a r a t ion of L-2-[4′-(E t h ylca r b a m oyl)p h en yl]-N-
a cetylglycin e (L-1). N-Acetyl-L-(4′-aminophenyl)glycine (2)3
(0.651 g, 3.39 mmol) was dissolved in a solution of potassium
carbonate (0.430 g, 3.12 mmol) in 1:1 aqueous dioxane. Ethyl
chloroformate (0.35 mL, 3.67 mmol) was added dropwise with
stirring at room temperature. After 15 min, TLC indicated
completed reaction. Water (10 mL) was added, and the reaction
mixture was extracted twice with ether. Acidification with 5%
HCl followed by extraction with ethyl acetate gave a yellow
oil, which was crystallized from ethyl acetate to give a white
addition, the assigned 13C resonance of the carbamoyl
carbonyl carbon of giganticine appeared at an unusually
downfield position (168.5 ppm), while most carbamoyl
carbonyl carbons, including that of L-1, resonate at much
higher field (154.3 ppm for L-1). These spectral differences
are too much to be accounted for by the different solvents
and the field strength of NMR spectrometer used.5 Full
assignments of the 1H and 13C NMR spectra of L-1 were
assisted by HMQC, HMBC, and NOESY experiments and
are fully consistent with the proposed structure.
Giganticine is a relatively nonpolar compound (TLC on
silica gel G, Rf 0.48, hexane-diethyl ether, 11:9; Rf 0.23,
acetone-petroleum ether, 1:4), and it can be extracted from
the CHCl3-soluble crude extract of the root bark of C.
gigantea using petroleum ether.1 Synthetic L-1 is a much
more polar compound, and therefore it scarcely moved on
TLC under the same conditions. Melting points of the two
compounds are also different (giganticine, 159-162 °C; L-1,
209-210 °C). On the basis of the above differences in
spectroscopic data and physical properties it is quite likely
that the two compounds are different.
Another independent synthesis of 1 was also investigated
(Scheme 2). Methyl 4-nitrophenylacetate (3) was treated
with isopentyl nitrite in the presence of methanolic sodium
methoxide6 to give the crystalline hydroxyimino ester (4)
in 57% yield. Reduction of both the nitro group and the
hydroximino function was carried out in one step using zinc
dust in acetic acid to give the diamino ester (5), which was
further acetylated using a limited amount of acetic anhy-
dride in the presence of N,N-diisopropylethylamine to give
the N2-acetylated amino ester (6) in 46% yield. Treatment
of 6 with ethyl chloroformate in the presence of N,N-
diisopropylethylamine gave the methyl ester (8) in 82%
yield. Saponification gave DL-1. Synthetic DL-1 and L-1
obtained from two independent methods displayed identical
1H and 13C NMR spectra and other physical characteristics,
except for melting point and optical rotation. These data
are, however, distinctively different from those of the
unspecified enantiomer of giganticine as reported by Pari
et al.1 It is therefore necessary to conclude that the
structure of giganticine is unlikely to be 1, and further work
should be done in order to disclose the true identity of
giganticine.7
22
crystalline solid (0.332 g, 35% yield): mp 209-210 °C; [R]D
+153.6° (c 0.5, DMF); IR (KBr) νmax 3500 (OH, NH), 3300 (NH),
1700 (CdO), 1600, 1500, 1230 cm-1 1H and 13C NMR, see
;
Table 1; EIMS (solid probe, 70 eV) m/z 280 [M+], 262, 236,
193, 165, 147. anal. C 55.22%; H 5.91%; N 9.72%, calcd for
C
13H16N2O5, C 54.93%, H 5.62%, N 9.85%.
P r ep a r a tion of DL-2-[4′-(Eth ylca r ba m oyl)p h en yl]-N-
a cetylglycin e (DL-1). A suspension of methyl hydroxyimino-
4-nitrophenylacetate (4)6 (1.00 g, 4.46 mmol) in 50% aqueous
acetic acid was treated with excess zinc dust at 5 °C for 1 h.
After the reaction was judged complete by TLC, the insoluble
matter was removed by filtration, and the solution was
adjusted to pH 8 with concentrated aqueous ammonia solution
followed by extraction with dichloromethane. The solvent was
evaporated to give the diamino ester (5) as a brown oily
residue. This was taken up in dichloromethane and treated
with acetic anhydride (0.28 mL, 2.94 mmol) in the presence of
excess N,N-diisopropylethylamine. After 30 min at room
temperature, the solvent was removed, and the residue was
dissolved in 10% aqueous HCl and extracted several times with
ethyl acetate to remove the diacetylated product (7). The
aqueous phase was made basic by addition of solid NaHCO3
and extracted with ethyl acetate. Evaporation of the solvent
gave the N2-monoacetylated product (6) as an oil (300 mg, 46%
2 steps). Treatment of 6 (220 mg, 1.35 mmol) with ethyl
chloroformate (0.14 mL, 1.50 mmol) and N,N-diisopropylethyl-
amine (0.25 mL, 1.50 mmol) in dichloromethane for 30 min at
room temperature followed by chromatography on silica gel
eluting with 60-80% ethyl acetate-hexane gave methyl ester
8 as a light yellow solid (0.327 g, 82% yield): 1H NMR (DMSO-
d6) 1.22 (3H, t, J ) 7 Hz, H-2′′), 1.87 (3H, s, CH3CO), 3.59
(3H, s, CH3 ester), 4.07 (2H, q, J ) 7 Hz, H-1′′), 5.27 (1H, d, J
) 7 Hz, H-2), 7.25 (2H, d, J ) 8 Hz, H-2′ and H-6′), 7.43 (2H,
d, J ) 8 Hz, H-3′ and H-5′), 8.60 (1H, d, J ) 7 Hz, NH-2), 9.67
(1H, s, NH-4′).
Aqueous NaOH (5%, 2.0 mL) was added to a solution of the
methyl ester 8 (0.258 g, 0.88 mmol) in methanol (5 mL) and
the reaction stirred at room temperature for 15 min. After
evaporation of the solvent, the residue was dissolved in H2O
and acidified to pH 2 with concentrated HCl. DL-1 precipitated
as a light yellow crystalline solid, which was collected by
filtration and dried under vacuum (0.196 g, 80%): mp 180-
182 °C; 1H NMR (DMSO-d6) 1.22 (3H, t, J ) 7 Hz, H-2′′), 1.86
(3H, s, CH3CO), 4.10 (2H, q, J ) 7 Hz, H-1′′), 5.21 (1H, d, J )
7.5 Hz, H-2), 7.26 (2H, d, J ) 8 Hz, H-2′ and H-6′), 7.42 (2H,
d, J ) 8 Hz, H-3′ and H-5′), 8.50 (1H, d, J ) 7.5 Hz, NH-2),
9.64 (1H, s, NH-4′); 13C NMR (DMSO-d6) 14.8, 22.6, 56.3, 60.6,
118.8, 128.7,131.6, 139.7, 154.3, 169.9,172.9.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. The optical rotation
was determined on a Perkin-Elmer 341 polarimeter. IR spectra
were recorded on a Nicolet Impact 410 spectrophotometer.
Spectra of solid samples were recorded as KBr pellets. Routine
1H NMR spectra were obtained on a Bruker ACF 200 operating
at 200 MHz (1H) and 50.28 MHz (13C). 2D NMR experiments
Ack n ow led gm en t. We acknowledge the financial support
from the Department of Chemistry, Faculty of Science, Chu-
lalongkorn University, the Biodiversity Research and Training
Program (BRT), BIOTEC/NSTDA (to T.V.), and DPST scholar-
ship (to C.S.). We also thank Dr. Khanit Suwanborirux

1116 J ournal of Natural Products, 2001, Vol. 64, No. 8
Notes
(5) L-1 is insoluble in CDCl3; therefore direct comparison of the spectra
in the same solvent cannot be made.
(6) Herbert, R. B.; Knaggs, A. R. J . Chem. Soc., Perkin Trans. 1 1992,
109-114.
(7) Attempts to isolate giganticine from the root bark of C. gigantea
collected in Thailand were unsuccessful since 1H NMR analysis of
the crude and partially purified extracts according to the method
described by Pari et al (ref 1) showed no sign of the compound.
(Faculty of Pharmaceutical Sciences, Chulalongkorn Univer-
sity) for recording some NMR spectra.
Refer en ces a n d Notes
(1) Pari, K.; Rao, P. J .; Devakumar, C.; Rastogi, J . N. J . Nat. Prod. 1998,
61, 102-104.
(2) Friis, P.; Kjaer, A. Acta Chem. Scand. 1963, 17, 2391-2394.
(3) Holdrege, C. T. U.S. Patent 3,479,339, 1969.
(4) Atkinson, R. N.; Moore, L.; Tobin, J .; King, S. B. J . Org. Chem. 1999,
64, 3467-3475.
NP010046L