Isolation and biomimetic synthesis of (±)-Guajadial B, a novel meroterpenoid from Psidium guajava

Article abstract of DOI:10.1021/ol302849v

(±)-Guajadial B (1), an unusual humulene-based meroterpenoid, was isolated as a racemate from the leaves of Psidium guajava, collected from Vietnam. The structure of this novel secondary metabolite was established on the basis of extensive analysis of NMR

Full text of DOI:10.1021/ol302849v

ORGANIC
LETTERS
2012
Vol. 14, No. 23
5936–5939
Isolation and Biomimetic Synthesis
of (()-Guajadial B, a Novel Meroterpenoid
from Psidium guajava
Yuan Gao,†,‡,§ Gang-Qiang Wang,† Kun Wei,† Ping Hai,‡ Fei Wang,†,‡ and Ji-Kai Liu*,†
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming
Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic
of China, BioBioPha Co. Ltd., Kunming 650201,
People’s Republic of China, and Graduate University of Chinese Academy of Sciences,
Beijing 100049, People’s Republic of China
jkliu@mail.kib.ac.cn
Received October 17, 2012
ABSTRACT
(()-Guajadial B (1), an unusual humulene-based meroterpenoid, was isolated as a racemate from the leaves of Psidium guajava, collected from
Vietnam. The structure of this novel secondary metabolite was established on the basis of extensive analysis of NMR spectra and confirmed by
biomimetic synthesis in a domino three-component coupling reaction.
Psidium guajava L. (Myrtaceae) is an evergreen shrub
grown in tropical and subtropical regions as a food and is
also an indigenous medicinal plant used for the treatment
of inflammatory, diabetes, hypertension, wounds, pain,
fever, vomiting, and diarrhea.1 Our previous studies on the
leaves of P. guajava led to the isolation of a rare caryo-
phyllene-based meroterpenoid guajadial.2 Subsequently,
several structurally novel merosesquiterpenoids were
isolated from the same plant.3,4 Recent studies by Thompson
et al. have verified the proposed biomimetic approach of
guajadial via a hetero-DielsꢀAlder reaction between car-
yophyllene and an o-quinone methide conducted in water
as a universal biological solvent.5 Our efforts in this area
have been continuing, and we would now like to report
the isolation, structure elucidation, and biomimetic synth-
esis of another unusual humulene-based meroterpenoid
(()-guajadial B (1) from P. guajava.
Leaves of P. guajava (15 kg) were collected from south of
Vietnam and extracted with MeOH at rt. After filtra-
tion, the methanolic extract was evaporated under reduced
pressure to obtain a residue (ca. 1400 g), which was frac-
tionized by silica gel column chromatography using
petroleum ether containing an increasing amount of
acetone. Successive purification of 2.5 g of the fraction
eluted with petroleum ether/acetone (100:1) by Sephadex
† Kunming Institute of Botany.
‡ BioBioPha Co. Ltd.
§ Graduate University of Chinese Academy of Sciences.
ꢀ
(1) (a) Gutierrez, R. M.; Mitchell, S.; Solis, R. V. J. Ethnopharmacol.
2008, 117, 1–27. (b) Jajrj, P.; Khoohaswan, P.; Wongkrajang, Y.;
Peungvicha, P.; Suriyawong, P.; Saraya, M.; Ruangsomboon, O.
J. Ethnopharmacol. 1999, 67, 203–212. (c) Shu, J. C.; Liu, J. Q.; Chou,
G. X.; Wang, Z. T. Chin. Chem. Lett. 2012, 23, 827–830.
(2) Yang, X. L.; Hsieh, K. L.; Liu, J. K. Org. Lett. 2007, 9, 5135–5138.
(3) Fu, H. Z.; Luo, Y. M.; Li, C. J.; Yang, J. Z.; Zhang, D. M. Org.
Lett. 2010, 12, 656–659.
(4) Shao, M.; Wang, Y.; Liu, Z.; Zhang, D. M.; Cao, H. H.; Jiang,
R. W.; Fan, C. L.; Zhang, X. Q.; Chen, H. R.; Yao, X. S.; Ye, W. C. Org.
Lett. 2010, 12, 5040–5043.
(5) Lawrence, A. L.; Adlington, R. M.; Baldwin, J. E.; Lee, V.; Kershaw,
J. A.; Thompson, A. L. Org. Lett. 2010, 12, 1676–1679.
r
10.1021/ol302849v
Published on Web 11/19/2012
2012 American Chemical Society

LH-20 (CHCl3/MeOH, 1:1) and preparative HPLC on a
ZORBAX SB-C18 column (90%f95% CH3CN in H2O
and 0.1% formic acid over 5 min followed by 95% CH3CN
in H2O and 0.1% formic acid to 17 min, 20 mL/min, 280
nm) afforded 6 mg of guajadial B (1, tR = 9.2 min).
Table 1. 1H and 13C NMR Spectroscopic Data for (()-1 in
CDCl3
no.
δ
H (J in Hz)
δC
1R
1β
2
2.04 (dd, 12.9, 11.8)
1.64 (dd, 12.9, 4.1)
4.52 (dd, 11.8, 4.1)
41.3 (t)
123.0 (d)
136.3 (s)
37.4 (t)
3
4R
4β
5R
5β
6
1.56 (dd, 13.1, 6.9)
0.58 (dd, 13.1, 11.0)
1.36 (dd, 14.9, 11.0)
1.42 (ddd, 14.9, 7.4, 6.9)
2.15 (dd, 10.1, 7.4)
30.7 (t)
43.4 (d)
85.2 (s)
42.4 (t)
7
8R
8β
9
2.36 (dd, 15.0, 9.3)
2.66 (ddd, 15.0, 1.4, 1.0)
5.18 (ddd, 16.0, 9.3, 1.4)
5.14 (dd, 16.0, 1.0)
119.3 (d)
143.1 (d)
38.6 (s)
Compound (()-1 was obtained as an amorphous pow-
der. The ion at m/z 474.2417 (calcd 474.2406) in its high-
resolution EI mass spectrum gave a molecular formula
10
11
12
13
14
15
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
50-OH
70-OH
1.44 (s)
16.7 (q)
of C30H34O5, which was in accordance with the 1H and 13
C
1.23 (s)
19.9 (q)
NMR data (Table 1). The IR spectrum suggested the
presence of hydroxyl (3431cmꢀ1) and conjugated carbonyl
(1634 cmꢀ1) functionalities. The UV spectrum of (()-1
showed the absorption maxima at 276 and 335 (sh) nm.
Analysis of the 1H NMR data revealed signals due to two
H-bonded phenolic hydroxyls at δH 13.48 and 13.11 (each
1H, s), two aldehydes at δH 10.10 and 10.07 (each 1H, s), a
monosubstituted benzene ring at δH 7.16ꢀ7.28 (5H, m),
and four methyls at δH 1.05, 1.05, 1.23, 1.44 (each 3H, s).
The 13C NMR (DEPT) spectrum showed a total of 30 car-
bon signals, including two aldehyde resonances at δC 192.2
(d) and 191.6 (d); signals for a monosubstituted phenyl at
δC 128.3 (4 ꢁ d) and 126.5 (d); a set of carbons due to a
hexasubstituted aromatic ring at δC 105.7 (s), 163.2 (s),
104.2 (s), 168.3 (s), 104.2 (s), and 169.3 (s); four methyls at
δC 16.7 (q), 19.9 (q), 23.9 (q), and 29.8 (q); signals ascribed
to two double bonds at δC 119.3 (d), 123.0 (d), 136.3 (s),
and 143.1 (d); and eight remaining aliphatic carbons.
Detailed analysis of its 2D NMR spectra and comparison
of the 1D NMR data with those of (()-2 resulted in un-
ambiguous assignment of all 1H and 13C NMR signals as
shown in Table 1.
1.05 (s)
23.9 (q)
1.05 (s)
29.8 (q)
3.60 (d, 10.1)
44.7 (d)
105.7 (s)
163.2 (s)
104.2 (s)
168.3 (s)
104.2 (s)
169.3 (s)
144.6 (s)
128.3 (d)
128.3 (d)
126.5 (d)
128.3 (d)
128.3 (d)
192.2 (d)
191.6 (d)
7.19 (m)
7.26 (m)
7.19 (m)
7.26 (m)
7.19 (m)
10.10 (s)
10.07 (s)
13.48 (s)
13.11 (s)
3,5-diformylbenzyl phloroglucinol was coupled with a
humulene moiety via a C-6ꢀC-10 bond as evidenced by
1
1Hꢀ H COSY cross-peaks between H-6 and H-5/H-10. Ac-
cording to the molecular formula information, it is plausible
to deduce that the oxygen atom leftover was to bridge C-7 at
δC 85.2 and C-30 at δC 163.2, to form a six-member ring.
In the HMBC spectrum (Figure 1), cross-peaks from
H-10 to C-20, C-30, C-70, C-80, C-90, and C-130 implied the
existence of a 3,5-diformylbenzyl phloroglucinol moiety
(1a), which was confirmed by careful comparison of chem-
ical shift values with those of guajadial,2 a novel 3,5-
diformylbenzyl phloroglucinol-coupled sesquiterpenoid
mentioned earlier. The HSQC spectrum established the
connectivity of the protons and the C-atoms, and the
1
1Hꢀ H COSY correlations between H-1 and H-2, H-5
and H-4/H-6, and H-9 and H-8/H-10 revealed the presence
of three spin systems (C-1/C-2, C-4/C-5/C-6, and C-8/C-9/
C-10) (Figure 1). A series of HMBC cross-peaks from
C(3)-Me to C-2, C-3, and C-4; from C(7)-Me to C-6, C-7,
and C-8; and from C(11)-Me to C-10, C-11, and C-1 allowed
the construction of a basic structural unit of humulene (1b).
Significant HMBC correlations from H-10 to C-5, C-6, and
C-7 and from H-6 to C-10 and C-80 confirmed that the
1
Figure 1. Key 1Hꢀ H COSY and HMBC correlations of (()-1.
The relative stereochemistry for (()-1 was establishedby
analysis of its ROESY data (Figure 2) and proton coupling
Org. Lett., Vol. 14, No. 23, 2012
5937

constants. Key correlations between H-10 and H3-13 and
between H-6 and the aromatic protons revealed the R-axial
orientation for H-10 and H3-13 and β-axial orientation
forH-6. Theseobservationsareingood agreement withthe
Scheme 1. Retrosynthetic Approach to (()-Guajadial B (1)
0
coupling constants (J1 ꢀ6 = 10.1 Hz) which indicated a
trans diaxial relationship for H-10 and H-6, as well as with
the chemical shift value of H-4β at δH 0.58 (the noticeable
upfield shift being due to the shielding by the aromatic
ring current of 10β-phenyl). The ROESY correlations of
H3-12TH-1R and H-2TH-4β supported the E-geometry
of the C-2/C-3 olefin. The E-geometry of C-9/C-10 olefin
was consistent with the large coupling constants ob-
served (J9ꢀ10 = 16.0 Hz) as well as with ROESY correlations
(H-9TH3-14/H3-15 and H-10TH-8R). In Figure 2, the
3D-presentation of (()-1 originated from the conforma-
tion with minimized energy by MM2 calculation using
ChemBio3D software. Conformationally, key ROESY
correlations of H-6TH-9/H-2 and H-10TH3-12 demon-
strated that H-6, H-9, and H-2 were on the same side, while
H-10 and H3-12 were on the opposite side as presented in
Figure 2. This secondary metabolite occurs as a racemate,
as evidenced by the lack of optical activity. The racemic
nature was also supported by subsequent HPLC analysis
of (()-1 on a chiral phase column, in which two distinct
chromatographic peaks appeared with a ratio of 1:1.
Table 2. Condition Screen for the Biomimetic Synthesis of
(()-Guajadial B (1)
Figure 2. Key ROESY correlations of (()-1.
yielda (%)
To confirm our proposed structure of (()-guajadial B
(1),6 the biomimetic synthesis of (()-1 was designed based
on a hetero-DielsꢀAlder reaction strategy. Our retrosyn-
thetic analysis of (()-1 was shown in Scheme 1; a [4 þ 2]
cycloaddition between natural R-humulene 6 and the
o-quinone methide 5 was enlisted to deliver (()-guajadial
B (1) in a biomimetic manner. In turn, the o-quinone
methide 5 was envisioned to arise from a Knoevenagel con-
densation7 between diformylphloroglucinol 3 and benzal-
dehyde 4. For synthetic efficiency and brevity, the prepara-
tion of the o-quinone methide 5 and subsequent biomimetic
[4 þ 2] cycloaddition could be performed in one pot.
entry
conditions
satd. NaCl
t (°C)
time
1
2
1
2
3
100
100
100
24 h
24 h
24 h
24
34
31
8
b
5% PTS(aq)
11
9
5% PTS(aq)
excess 6 (10 equiv)
AcOH þ NaOAc
(0.1 equiv)
4
5
6
80
80
80
24 h
72 h
120 h
39
43
45
14
18
19
AcOH þ NaOAc
(0.1 equiv)
AcOH þ NaOAc
(0.1 equiv)
a HPLC yields with MeOH/H2O condition. b PTS: PEG-600/R-To-
copherol-based diester of Sebacic acid.
Indeed, a similar strategy had been applied in the
biomimetic synthesis of guajadial by Thompson et al.5
However, the situation with the biomimetic approach is
obscured bythe issue thatthere are three endocyclicdouble
bonds in the 11-membered sesquiterpene ring, each of
which is potentially capable of reacting with o-quinone
methide 5. It is known, however, that the trisubstituted
double bond situated furthest from the gem-dimethyl
(6) (()-Guajadial B (1): white powder; [R]2D5 0 (c 0.20, CHCl3); UV
(MeOH) λmax: 276, 335 (sh) nm; IR (KBr) νmax 3441, 2958, 2927, 2858,
1634, 1442, 1383, 1303, 1191, 1157, 1047, 843, 756, 699, 608 cmꢀ1; EI-
MS: m/z 474 [M]þ (100), 405 (45), 391 (62), 323 (15), 309 (25), 279 (20),
271 (58), 233 (21), 167 (25), 149 (52); HR-EI-MS: m/z 474.2417 (calcd for
C30H34O5, 474.2406).
(7) Knoevenagel, E. Ber. Dtsch. Chem. Ges. 1898, 31, 2596–2619.
5938
Org. Lett., Vol. 14, No. 23, 2012

group of R-humulene 6 is the more reactive alkene; due
to the steric hindrance, we believe the syn-addition of
o-quinone methide 5 must involve the trisubstituted olefin.
To determine whether this strategy is chemically feasible
for the synthesis of (()-guajadial B (1), preparation of
diformylphloroglucinol 3, a precursor of o-quinone methide
5, was required. The precursor 3 was facilely synthesized in
82% yield by using Hintermann’s conditions.8
under conventional heating at 80 °C for 24 h resulted in a
yield of 53% with 14% (()-2 (Table 2, entry 4). Extending
the heating period to 3 days gave a promising yield of 61%
with 18% (()-2 (Table 2, entry 5). However, when we
extended the period by an additional 2 days, no significant
improvement in yield was observed at the expense of
reaction time (Table 2, entry 6). Subsequent column
chromatography was followed by HPLC purification of
the cleanest fractions, resulting in an unoptimized 27%
isolated yield of (()-guajadial B (1), identical by NMR,
TLC, and HPLC comparison with the natural product,
and a 6% yield of (()-2. Consistent with their nonenzy-
matic route, both products occur as racemates as evi-
denced by the lack of optical activity and by HPLC
analyses on a chiral phase column.
In summary, we have isolated and characterized the
humulene-based meroterpenoid (()-guajadial B (1) from
P. guajava L. We revealed that when using water as a
universal biological solvent, the naturally occurring (()-1
is the favored monoadduct of R-humulene 6 with o-qui-
none methide 5 generated under nonenzymatic conditions
at elevated temperatures. This offers positive evidence that
a similar route may occur to provide (()-1 biosynthetically.
With diformylphloroglucinol 3 in hand, various condi-
tions were investigated to determine the feasibility of effect-
ing its addition to (()-1. Our initial attempt to synthesize
(()-1 was carried out by using water as the reaction
medium, and NaCl was added due to its dehydrating nature.9
Heating the three-compound mixture in saturated brine for
24 h afforded the desired (()-guajadial B (1) in a 24% yield,
together with another compound which was later assigned as
(()-2, an C-10 epimer of (()-1, in 8% yield (Table 2, entry 1).
Considering the unequal dispersal of the reactants in
water, surfactant has been used to decrease the hetero-
geneity.10 In an aqueous solution of 5 wt % PEG-60/R-
Tocopherol-based diester of Sebacic acid (PTS) as a non-
ionic surfactant, a better chemical yield of 45% with 11%
(()-2 was achieved (Table 2, entry 2). The same procedure
was applied except that 10 equiv of 6 were added, resulting
in a total yield of 40% with 9% (()-2 (Table 2, entry 3).
Higher efficiency in the synthesis of (()-1 was sought by
using the conditions of Thompson et al.5 Treatment of the
mixture in the presence of sodium acetate in acetic acid
Acknowledgment. The authors acknowledge the National
Basic Research Program of China (973 Program, 2009-
CB522300) and the National Natural Science Founda-
tion of China (30830113, U1132607).
Supporting Information Available. Synthetic proce-
dures and analytic data. This material is available free
of charge via the Internet at http://pubs.acs.org.
(8) Dittmer, C.; Rabbe, G.; Hintermann, L. Eur. J. Org. Chem. 2007,
5886–5898.
(9) He, H.; Pei, B. J.; Chou, H. H.; Tian, T.; Chan, W. H.; Lee,
A. W. M. Org. Lett. 2008, 10, 2421–2424.
(10) Doncaster, J. R.; Ryan, H.; Whitehead, R. C. Synlett 2003, 5,
651–654.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 23, 2012
5939