Is 2,3,4,5-tetramethoxybenzoyl chloride a natural product?

Article abstract of DOI:10.1021/np200095q

The title compound, which was reported to be a constituent of the fruiting body of the fungus Antrodia camphorata, has been synthesized. The reactivity and spectroscopic properties of the synthetic material do not match those of the natural product. There

Full text of DOI:10.1021/np200095q

NOTE
pubs.acs.org/jnp
Is 2,3,4,5-Tetramethoxybenzoyl Chloride a Natural Product?
Kathryn A. Punch, Emilio L. Ghisalberti, and Matthew J. Piggott*
School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Western Australia, 6009
ABSTRACT: The title compound, which was reported to be a constituent of the fruiting body of the fungus
Antrodia camphorata, has been synthesized. The reactivity and spectroscopic properties of the synthetic material
do not match those of the natural product. There is currently insufficient information for a definitive structural
reassignment.
n 2007, 2,3,4,5-tetramethoxybenzoyl chloride (2) (Scheme 1)
Scheme 1a
I
was purportedly isolated from the hexane extract of the fruiting
body of Antrodia camphorata, a highly valued medicinal fungus
from Taiwan.1 This was surprising for two reasons. First, the
unprecedented discovery of an acid chloride natural product had
been published without comment. Second, the acid chloride not
only was biosynthesised in an aqueous environment but was
stable enough to survive the extraction and purification process,
including multiple chromatographic separations using silica gel.
This was an acid chloride unlike any other in our experience!
Even the highly sterically hindered 2,4,6-tri-tert-butylbenzoyl
chloride undergoes rapid hydrolysis.2
The structural elucidation of the natural product by Chen
et al. was based upon detailed spectroscopic studies, including
2D NMR experiments, low- and high-resolution mass spectra,
and the product of methanolysis. While the spectroscopic
data could not be faulted at face value (in the absence of
authentic material), close examination of the other evidence
revealed several anomalies. Most immediately obvious was
the absence of the 37Cl isotope peaks from the low-resolution
electron impact ionization mass spectral data. Furthermore,
the molecular ion (containing 35Cl) was the base peak, and
there was no [M ꢀ Cl]þ fragment at m/z 225. The [M ꢀ Cl]þ
fragment gives rise to the base peak in the mass spectra of o-,
m-, and p-anisoyl chloride, 2,6-, 3,4-, and 3,5-dimethoxyben-
zoyl chloride, and all produce molecular ions of relatively low
abundance (26% at most, but generally less than 10%).3 Thus,
on the basis of the mass spectrum alone, it seemed highly
unlikely that the natural product was an acid chloride.
Treatment of the natural product with pyridine/MeOH
resulted in substitution, affording a product that was assigned
as the methyl ester 3.1 Again, at face value the characterization
data for this compound seemed appropriate, but they were not
compared with those already in the literature; methyl 2,3,4,5-
tetramethoxybenzoate (3) has previously been isolated from
Relhania acerosa.4 The frequency of the carbonyl stretch absorp-
tion in the IR spectrum, the 1H NMR data, and the EIMS mass
spectra differ significantly between the publications, as detailed in
Table 1. Thus, it appeared that the methanolysis product
characterized by Chen et al. had been structurally misassigned
and, ergo, so had the natural product.
a Reagents and conditions: (a) (COCl)2, DMF, DCM; 2 ꢁ Kugelrohr
dist. (38%); (b/c) 1. SOCl2, 2. MeOH, pyridine (80% over two steps);
(d) DCC, DCM.
The anomalies discussed above prompted us to synthesize 25
and the corresponding methyl ester 3. The acid chloride 2 was
simply prepared from the known benzoic acid 16 by treatment with
oxalyl chloride and catalytic N,N0-dimethylformamide (Scheme 1).
In contrast to the robust natural product described by Chen et al.
(the UV spectrum was acquired in MeOH), we found 2 to be
extremely moisture sensitive. Indeed, it was exceedingly difficult to
obtain a clean 13C NMR spectrum of 2 due to rapid hydrolysis
resulting from adventitious water in CDCl3. The hypersensitivity of
2 to hydrolysis presumably results from the electron-donating o/p-
methoxy groups, which facilitate an SN1 mechanism by stabilization
of the intermediate acylium ion.7
The spectroscopic and MS data for synthetic 2 are presented
in Table 2. As anticipated, the NMR data differ significantly
from those reported for the natural product. As expected, the
[Mꢀ Cl]þ fragment gave rise to the base peak in the mass spectrum
Received: January 30, 2011
Published: April 14, 2011
r
Copyright
2011 American Chemical Society and
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dx.doi.org/10.1021/np200095q J. Nat. Prod. 2011, 74, 1348–1350
American Society of Pharmacognosy
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Journal of Natural Products
NOTE
Table 1. Spectroscopic and MS Data for 3 from Various
Sources
Chen et al.1
Bohlmann et al.4
synthetic
IR (cmꢀ1
1H NMR (δ, ppm)
)
1714
7.03a
1735
7.12b
7.10c
3.91
3.96
3.96
Figure 1
3.91
3.91
3.91
3.90
3.90
3.90
of the synthetic material, and monoisotopic molecular ions were
detected, albeit at very low abundance.
3.90
3.88
3.88
3.89
3.87
3.86
To help confirm the structure of the acid chloride 2 and allow
comparison of the methyl ester 3 with literature reports, the
former was subjected to the methanolysis conditions used by
Chen et al. Thus, treatment of 2 with 1:1 pyridine/MeOH gave
the corresponding ester 3, which differed significantly from the
methanolyzed natural product, but was virtually identical in all
respects to that isolated by Bohlmann et al.4 (Table 1). Clearly,
2,3,4,5-tetramethoxybenzoyl chloride (2) is not the natural
product isolated by Chen et al. This evidence also indicates that
the natural product is not a derivative of 2,3,4,5-teramethoxy-
benzoic acid 1. Nevertheless, we considered two such derivatives
with the aim of potentially identifying functional groups that
match the chemical reactivity and at least some of the spectro-
scopic properties of the natural product.
EIMS [m/z (%)]d
256 (100)
241 (54)
225 (51)
210 (57)
256 (100)
256 (100)
241 (45)
225 (28)
210 (4)
198 (26)
197 (1)
225 (51)
197 (65)
a Recorded at 400 MHz. b Recorded at 270 MHz. c Recorded at
500 MHz. d Not all reported data are shown.
Table 2. Spectroscopic and MS Data for 2 from Various
Sources
Chen et al.1a
synthetic
Although an anhydride is likely also too reactive to be a natural
product, we hypothesized that perhaps a benzoic acid was
isolated and, in the presence of catalytic acid during the drying
process, afforded the anhydride. Accordingly, anhydride 4
(Scheme 1) was prepared by heating 1 with DCC. Both the
carbonyl carbons (153.5 ppm) and aromatic protons (6.47 ppm)
of 4 resonate significantly upfield of the corresponding signals in
the natural product, ruling out an anhydride as the reactive
functional group in the latter.
IR (CdO, cmꢀ1
)
1768
7.13
1775
7.30
1H NMR (ppm)
3.93
4.01
3.92
3.91
3.91
3.90
3.89
3.89
13C NMR (ppm)
168.1
151.0
148.2
147.9
146.5
116.5
109.7
62.4
163.5
149.7
149.2
148.8
147.3
121.5
110.7
62.0
Methanolysis of the primary benzamide 5 (Figure 1) is
plausible, and it is also possible that the resonances due to the
1
amide protons were overlooked in the H NMR spectrum.
However, 5 is known,6 and while the carbonyl carbon resonates
in the appropriate region (166.6 ppm), the aromatic proton
(6.75 ppm) again resonates significantly upfield of the aryl signal
in the 1H NMR spectrum of the natural product. Furthermore,
the amide carbonyl absorbs much lower energy IR radiation than
the carbonyl group in the natural product.
61.2
61.5
61.1
61.3
56.3
56.4
We have been unable to propose an alternative structure that
might be consistent with the reported physical and chemical
properties. We initially considered a ring-chlorinated ester such
as 6, as the chlorine atom is much less readily lost in the mass
spectra of chlorobenzenes compared to acid chlorides, and in
principle, nucleophilic aromatic substitution with MeOH is
possible (although unlikely). However, the natural product gives
rise to four low-field resonances in the 13C NMR spectrum,
corresponding to four methoxy-substituted aromatic carbons,
and one such resonance would be absent in 6 (or one of its
regioisomers).
EIMS [m/z (%)]
262 (0.5)
260 (1.8)
260 (100)
245 (64)
231 (16)
229 (54)
225 (100)
217 (28)
227 (39)
217 (56)
219 (19)
213 (32)
Another structure considered was the tropolone 7, which
should be susceptible to nucleophilic substitution with MeOH
and might exhibit appropriate NMR chemical shifts. However,
the carbonyl group of this highly conjugated ketone would be
expected to absorb below 1700 cmꢀ1 in the IR spectrum. Indeed
DFT calculations predict a CdO stretch absorption at
1674 cmꢀ1, in contrast to the high-energy absorption of the
natural product (1768 cmꢀ1).
209 (19)
195 (21)
202 (25)
173 (21)
171 (54)
167 (18)
a Not all reported IR and MS data are shown.
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Journal of Natural Products
NOTE
It is apparent that more information is required for a structural
reassignment. As a starting point, the presence of chlorine in the
natural product needs to be confirmed by observation of the 37Cl
isotope peaks in the mass spectrum. Our attempts to derive a
different molecular formula based on the accurate mass determi-
nation were fruitless.
(6) Meyers, A. I.; Flisak, J. R.; Aitken, R. A. J. Am. Chem. Soc. 1987,
109, 5446–5452.
(7) Bender, M. L.; Chen, M. C. J. Am. Chem. Soc. 1963, 85, 30–36.
(8) Gandy, M. N.; Piggott, M. J. J. Nat. Prod. 2008, 71, 866–868.
In conclusion, we have presented irrefutable evidence that a
natural product isolated from Antrodia camphorata has been
misassigned as 2,3,4,5-tetramethoxybenzoyl chloride (2). The
identity of the metabolite remains to be established.
’ EXPERIMENTAL SECTION
General experimental procedures have been published previously.8
Computational Modeling. Modeling was conducted using Spar-
tan 08 at the density functional theory 6-31G* level.
2,3,4,5-Tetramethoxybenzoyl Chloride (2). Oxalyl chloride
(61 mg; 0.47 mmol) was added to a stirred solution of 1 (110 mg,
0. 43 mmol) in 0.1% anhydrous DMF/dichloromethane (1.5 mL) at
0 °C under argon. The reaction mixture was stirred for 6 d, the volatiles
were evaporated, and the residual brown oil was distilled twice
(Kugelrohr, 100ꢀ150 °C, 45 mmHg), to give 2 as a pale pink oil, which
solidified on cooling (45 mg; 38%). See Table 2 for spectroscopic data.
Methyl 2,3,4,5-tetramethoxybenzoate (3). Thionyl chloride
(1 mL) was added to a stirred solution of 1 (59 mg; 0.25 mmol). After
30 min, a 1:1 pyridine/MeOHsolution (1 mL) was added, and stirring was
continued for 3 h. The solution was diluted with DCM (20 mL), washed
with a saturated CuSO4 solution (3 ꢁ 10 mL), dried, and evaporated to
give 3 as a colorless oil (50 mg; 80%). The 1H NMR spectrum (500 MHz,
CDCl3) matched that reported.4
2,3,4,5-Tetramethoxybenzoic Anhydride (4). DCC (10 mg,
0.050 mmol) was added to a solution of 1 (10 mg, 0.040 mmol) in
anhydrous DCM, and the reaction mixture was heated under reflux for
36 h. Filtration of the reaction mixture through three plugs of neutral
alumina and evaporation of the filtrate gave 4 as a white solid (11 mg,
60%): 1H NMR (500 MHz, CDCl3) δ 6.47 (s, 2H, ArH), 3.94 (s, 6H,
CH3O), 3.90 (s, 6H, CH3O), 3.86 (s, 6H, CH3O), 3.81 (s, 6H, CH3O);
13C NMR (125.8 MHz; CDCl3) δ 153.6 (CO), 150.2 (ArO), 147.4
(ArO), 144.2 (ArO), 143.1 (ArO), 126.9 (ArC), 104.1 (ArH), 62.6
(CH3O), 61.3 (2 ꢁ CH3O), 56.5 (CH3O).
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ61 8 6488 3170. Fax: þ61 8 6488 1005. E-mail:
piggott@cyllene.uwa.edu.au.
’ ACKNOWLEDGMENT
We thank Dr. A. Reeder for mass spectra. K.A.P. is the
recipient of an Australian Postgraduate Award.
’ REFERENCES
(1) Chen, J.-J.; Lin, W.-J.; Liao, C.-H.; Shieh, P.-C. J. Nat. Prod. 2007,
70, 989–992.
(2) Frey, J.; Rappoport, Z. J. Chem. Soc., Perkin Trans. 1 1997,
1395–1397.
(3) Wasada, N. , SDBS. National Institute of Advanced Industrial
Science and Technology (AIST).
(4) Bohlmann, F.; Jakupovic, J. Phytochemistry 1979, 18, 631–635.
(5) The acid chloride has been prepared previously but was treated
as a synthetic intermediate and was not characterized in any way: Ghosal,
S.; Chaudhuri, R. K.; Markham, K. R. J. Chem. Soc., Perkin Trans. 1
1974, 2538–2541.
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