Syntheses of Gibberellins A15 and A24, the Key Metabolites in Gibberellin Biosynthesis

Article abstract of DOI:10.1021/acs.joc.8b00876

Gibberellins (GAs) are essential phytohormones involved in numerous aspects of plant growth and development. Notably, the biochemistry and genetics of GA biosynthesis, which is associated with their endogenous regulation, have been largely resolved; however, a crucial unsolved question remains: the precise mechanism of the stepwise oxidation and subsequent removal of C-20 from C₂₀ precursors, leading to bioactive C₁₉ gibberellins, is still unresolved. To satisfy numerous requests from biologists, practical preparations of certain GAs that were isolated in miniscule quantities are highly demanded. Herein, we report the first practical syntheses of GA₁₅ and GA₂₄, the key C₂₀ metabolites in gibberellin biosynthesis, from commercially available GA₃. The protocols are robust and offer the capacity to produce GA₂₄ and GA₁₅ under gram scales in high overall yields and thus aid in further biological and related studies.

Full text of DOI:10.1021/acs.joc.8b00876

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Article
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Syntheses of Gibberellins A and A, the Key
Metabolites in Gibberellin Biosynthesis
David J.-Y. D. Bon, Lewis Norman Mander, and Ping Lan
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00876 • Publication Date (Web): 22 May 2018
Downloaded from http://pubs.acs.org on May 22, 2018
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Syntheses of Gibberellins A15 and A24, the Key
Metabolites in Gibberellin Biosynthesis
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David J.ꢀY. D. Bon‡, Lewis N. Mander*,§ and Ping Lan*,†
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†Department of Food Science and Engineering, Jinan University, Guangzhou, 510632, China
‡Department of Organic Chemistry, Faculty of Science, Palacky University, tř. 17. listopadu
1192/12, CZꢀ771 46 Olomouc, Czech Republic
§Research School of Chemistry, Institute of Advanced Studies, The Australian National
University, Canberra, ACT 2601, Australia
*Corresponding author
*Telephone: +86ꢀ20ꢀ85221367. Fax: +86ꢀ20ꢀ85224766.
Eꢀmail: lew.mander@anu.edu.au. (L. N. M.)
Eꢀmail: ping.lan@jnu.edu.cn. (P. L.)
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ABSTRACT
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Gibberellins (GAs) are essential phytohormones involved in numerous aspects of plant
growth and development. Notably, the biochemistry and genetics of GA biosynthesis which
is associated with their endogenous regulation has been largely resolved, however, a crucial
unsolved question remains: the precise mechanism of the stepwise oxidation and subsequent
removal of Cꢀ20 from C20ꢀprecursors leading to bioactive C19 gibberellins is still unresolved.
In order to satisfy numerous requests from biologists, practical preparations of certain GAs
that were isolated in miniscule quantities are highly demanded. Herein we report the first
practical syntheses of GA15 and GA24, the key C20 metabolites in gibberellin biosynthesis,
from commercially available GA3. The protocols are robust and offer the capacity to produce
GA24 and GA15 under gram scales in high overall yields and thus aid in further biological and
related studies.
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INTRODUCTION
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The gibberellins (GAs) are a large class of highly functionalized diterpenoids that are
commonly found throughout the plant kingdom where they serve as plant hormones.1,2 These
hormones regulate a variety of plant developmental processes, notably – the germination of
seeds, trichome formation, pollen maturation, and play a role in the commencement of
flowering.3 For instance, mutant plants that express lower levels of GA exhibit a dwarf and
lateꢀflowering phenotype, when this deficiency is supplemented with exogenous GA, normal
plant growth is restored.4 The profound effects of GAs on plants make them a valuable tool in
modern agriculture to increase crop yields.5,6 As a consequence, GAs are of considerable
agricultural significance, not surprisingly, they have been widely accepted throughout the
world as a useful agricultural chemical for the treatment of increasing numbers of fruit and
vegetable crops including grapes, citrus, apples, artichokes, tomatoes, rice and grains.7ꢀ9
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Since the first discovery of GA in the pathogenic fungus Gibberella fujikuroi, the
compound responsible for the “silly seedling” disease of rice,10 more than 136 GAs have
been identified from plants, fungi and bacteria.11 Among the known GAs, only a small
number of them, such as GA1 (1), GA3 (2), GA4 (3) and GA7 (4) are biologically active, and
they are analogous possessing a pentacyclic diterpenoid framework with a Cꢀ6 carboxylic
acid, Cꢀ3 hydroxyl functionality and exocyclic olefin (Figure 1).4 Meanwhile, many
nonbioactive GAs are present in plants as “dormant” precursors for the bioactive forms or are
in turn deactivated metabolites.12 The regulation of the GA levels is believed to be complex
given the numbers of GAs isolated from nature, which has received a great deal of attention.3
Figure 2 summarizes the brief biosynthesis of major bioactive GAs.12 It was accepted early
on that the biosynthesis of GA begins from entꢀkaurenoic acid (5) and through a
hydroxylation at Cꢀ7 (leading to entꢀ7αꢀhydroxykaurenoic acid 6) and subsequent ring
contraction with expulsion of Cꢀ7 the GA progenitor, GA12 aldehyde (7) is formed.12 In both
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processes, entꢀkaurenoic acid oxidase (KAO) plays a crucial role, in fact, this KAO also
converts the GA12 aldehyde into GA12 (8). The biosynthesis then diverges based on the
organism involved (plant, fungi or bacteria) however the pathways are analogous overall,
varying mainly by the stage, position and level of oxidation.2 Thus, GA12 is sequentially
converted to GA15 then to GA24 and GA9, by GA 20ꢀoxidase (GA20ox), and upon installation
of the Cꢀ3 hydroxyl group by GA 3ꢀoxidase (GA3ox), the bioactive GA4 is generated. In
parallel, GA12 is also a substrate for GA 13ꢀoxidase (GA13ox) for the production of GA53,
which is a precursor for GA1 (through the GA44→GA19→GA20 pathway) and GA3 (via the
GA20→GA5 pathway). In particular, GA biosynthesis is tightly regulated by those enzymes,
especially the GA20ox and GA3ox that catalyse the crucial steps in the biogenesis of active
GAs.13,14
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Figure 1. Chemical structures of bioactive gibberellins A1, A3, A4 and A7.
Although the chemistry, biochemistry and genetics of GA biogenesis has been largely
resolved,4,10,12,13 a crucial unsolved question remains: the precise mechanism of the stepwise
oxidation and subsequent removal of Cꢀ20 from C20ꢀprecursors (i.e., GA15 and GA44) leading
to bioactive C19 gibberellins is still unresolved, despite that these bioꢀtransformations have
been observed in many plants as well as cellꢀfree systems.15ꢀ17 Numerous biologists having
taken up this challenge were struggling to gain access to several C20ꢀprecursors, preferably
with incorporated isotopes. GA15 and GA24, the key C20ꢀprecursors to GA4, are of particular
interest to biologists. They have been isolated from various higher plants, though often in
miniscule quantities.1 Manifold efforts have been made to prepare those metabolites via
chemical synthesis. The first total synthesis of GA15 was accomplished by Nagata et al. in
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1971,18 albeit in a racemic form, moreover, this synthesis suffered from multiꢀstep procedure
and the starting materials were not readily available. Somei et al.19 reported the chemical
interconversion of enmein to GA15 in 1970, however, this method was lowꢀyielding and the
final purification was complex. The interconversions of gibberellins seem to be
advantageous, for instance, Ward et al.15 prepared the labelled GA15 from GA13 trimethyl
ester via an eightꢀsteps sequence, nevertheless, the GA13 has to be isolated from plants after
tedious procedures.
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KAO
KAO
KAO
13ox
H
H
7
H
H
OH
H
H
H
H
CO2H
CHO
CO2H
GA12 (8)
20ox
CO2H
CO2H
CO2H
ent-Kaurenoic
acid (5)
ent-7 -Hydroxy
Kaurenoic acid (6)
GA12 Aldehyde (7)
HO
HO
OHC
H
OH
OH
OH
20ox
20ox
H
H
H
H
CO2H
H
CO2H
H
CO2H
H
CO2H
CO2H
CO2H
CO2H
CO2H
GA19 (14)
GA15 (9)
GA44 (13)
GA53 (12)
open lactone
20ox
open lactone
20ox
CO2
OH
OH
OHC
H
O
H
O
H
O
H
3ox
20ox
CO2
H
HO
H
CO2H
H
H
CO2H
CO2H
CO2H
CO2H
O
O
O
GA24 (10)
GA20
(
15)
GA9 (11)
GA1 (1)
bioactive
3ox
3ox
OH
OH
O
O
O
H
3ox
H
H
H
H
HO
HO
H
CO2H
CO2H
CO2H
O
O
O
GA5 (16)
GA3 (2)
GA4 (3)
bioactive
bioactive
Figure 2. Biosynthetic route of bioactive gibberellins A1, A3 and A4 from entꢀkaurenoic acid. KAO: entꢀ
kaurenoic acid oxidase; 20ox: GA 20ꢀoxidase; 3ox: GA 3ꢀoxidase; 13ox: GA 13ꢀoxidase.
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In order to satisfy numerous requests for biosynthetic and related studies, herein we
report the first practical syntheses of GA15 and GA24, from GA3 which is now produced by
industrial fermentation at ton scale. The conversion of the C19 member GA3 to C20 ones GA15
and GA24 must contend with several difficulties: the introduction of Cꢀ20 into the very
hindered Cꢀ10 position on the concave αꢀface of the gibberellin molecule, and the elaboration
of an aldehyde group in a 1,3ꢀsynꢀdiaxial relationship to the Cꢀ19 carboxylic acid. By
utilizing the methodologies established over the past thirty years,2 we are now able to
complete the concise practical syntheses of GA15 and GA24.
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Scheme 1. Establishment of the C20 Framework from GA3.
MeI, K2CO3
Me2CO
18 °C, 20 h
MsCl
pyridine
0 to 18 °C, 13 h
OH
OH
OMs
O
H
O
O
H
H
H
H
94%
92%
HO
HO
MsO
H
CO2H
CO2Me
CO2Me
O
O
O
GA3 (3)
17
18
Li, NH3
THF/t-BuOH
–78 °C, 5 h
59%
Cu cat. 22
THF/cyclohexane
90 °C, 2 h
(COCl)2, DMF
Et2O/pyridine
0 to 18 °C, 24 h
1
1
10
10
2
H
H
H
20
53% over 3 steps
H
H
H
CO2Me
20 R = Cl
CO2Me
19
CO2Me
ROC
HO2C
O
23
CH2N2, Et2O
0 to 18 °C, 16 h
2. DMP
1. Li, NH3
THF/t-BuOH CH2Cl2/pyridine
21 R = CHN2
–78 °C, 5 h
0 °C, 5 h
KHMDS then O2
OHC
THF/DMF
0 °C, 3 h
77%
OHC
N
O
Cu
2
H
H
H
20
H
H
H
CO2Me
19
CO2Me
CO2Me
HO2C
MeO2C
O
24 (77% over 2 steps)
25
22
26
RESULTS AND DISCUSSION
As shown in Scheme 1, the synthesis begins with the use of GA3 (3), a commercially
available and biosynthetically abundant compound obtained as a microbial metabolite.20
From here standard esterification with methyl iodide under basic conditions, methyl
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gibberellate (17) was achieved in excellent yield (94%). At this point, for the synthesis of the
norꢀhydroxy gibberellins A15 and A24, both of the Cꢀ3 and Cꢀ13 hydroxyl groups of 17 have
to be removed. This was achieved by diꢀmesylation of the hydroxyl functionality leading to
dimesyl methyl gibberellate (18) followed by dissolving metal reduction.21 This dissolving
metal reduction is a synthetically powerful reaction serving to not only affect the 13ꢀhydroxyl
functionality but also deletion of the superfluous 3ꢀhydroxyl with isomerisation of the olefin,
expelling the lactone functionality to provide acid (19) in a respectable yield of 59%. The
rearrangement of the olefin is crucial for insertion of the extra carbon to the concave,
sterically hindered and preꢀquaternary carbon centre (Cꢀ10) and thus generation of C20 GAs
from their C19 congeners. Thus, the newlyꢀgenerated proximal carboxylic acid (19) was
converted into the diazoketone (21) via formation of the acid chloride (20) followed by
treatment of the latter with diazomethane. Upon treatment with the commercially available
copper (II) catalyst (22), the final carbon was added through carbene addition to the olefin
providing the corresponding cyclopropane (23) in 53% over 3 steps.22 Salicylateꢀderived
copper catalyst (22) was found to produce the most satisfactory results wherein minimal
formation of the CꢀH insertion side product at the Cꢀ2 position was observed. Employment of
another dissolving metal reduction23,24 exclusively resulted in cleavage of the less hindered
C1ꢀC20 bond in cyclopropane (23) to provide the desired ketone (24) with some overꢀ
reduced alcohols which were recycled through a subsequent oxidation step with DessꢀMartin
periodinane, thus affording the ketone (24) in 77% yield. Functionalization of the extremely
hindered Cꢀ20 position in ketone (24) as a prelude to oxidative fission had previously proved
to be very difficult.22 Herein we report a new and effective method for the oxidative cleavage
of the C19ꢀC20 bond and required formation of an enolate intermediate by using potassium
hexamethyldisilazide (KHMDS) in THF/DMF. The fission of this bond was then achieved
through treatment of the generated enolate with a steady stream of oxygen gas into the
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reaction mixture, yielding halfꢀacid (25) in a satisfactory yield (77%). Implementing the base
KHMDS to initiate the enolate formation was found to be safe and efficient even under an
atmosphere of oxygen. Due to serious lactol resonances, further confirmation of compound
(25) was achieved through derivatization with diazomethane so providing the methyl ester
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1
(26) which was not susceptible to lactol tautomer equilibrium effects,25,26 providing clear H
and 13C NMR spectra data.27
Scheme 2. Completion of the Syntheses of GA24 and GA15.
The elaboration of compound (25) to target GA24 (10) and GA15 (9) is outlined in
Scheme 2. Saponification of methyl ester (25) provided GA24 (10) in 87% yield, which again,
was treated with diazomethane so as to afford the dimethyl ester (26) for further structural
determination. With GA24 in hand, clean reduction with sodium borohydride delivered the
lactone GA15 (9) in high yield (92%). Surprisingly, although GA15 has been isolated from
various higher plants,1,28ꢀ32 as well as been prepared by some synthetic work,15,18,19 the full
NMR spectra data of this compound has not been reported. In current work, we are now able
to provide a complete set of characterization data for GA15 (9) by undertaking IR, MS
spectrometric and NMR spectroscopic analyses.
CONCLUSION
The protocols defined here are robust and offer the capacity to produce GA24 and GA15
under gram scales in high overall yields (13% and 14%, respectively). The strategies could
also be applied to pursue certain labelled GAs by using some labelled reagents (i.e., 14C
diazomethane) or by incorporating the terminal olefin protons at Cꢀ17 with deuterons.33 Given
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the versatile applications of GA3 in construction of structurally diverse compounds34 as well
as bioactive agents,35 the chemistry developed in this work provides valuable insights into the
reactivity of GA3.
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EXPERIMENTAL SECTION
Materials. Gibberellin A3 (>90%), methyl iodide (>99%), methanesulfonly chloride
(>99%), tertꢀbutyl alcohol (>99%), lithium metal (>99%), oxalyl chloride (>99%), bis(Nꢀtertꢀ
butylsalicylaldiminato)copper(II) [96%], potassium hexamethyldisilazide solution, Dessꢀ
Martin periodinane (97%), sodium borohydride (>99%), were all purchased from Sigmaꢀ
Aldrich (St. Louis, MO). Aluminumꢀbacked 0.2 mm thick silica gel 60 F254 plates for
analytical thin layer chromatography (TLC) as well as silica gel 60 (40–63 ꢀm) for flash
chromatographic separations were supplied by Merck (Darmstadt, Germany).
General Experimental Protocols. Proton (1H) and carbon (13C) NMR spectra were
recorded at 25 °C on a Bruker spectrometer (Billerica, MA) operating at 400 MHz for proton
and 100 MHz for carbon nuclei. For 1H NMR spectra, the residual protioꢀsolvent signals were
used as internal standards. Infrared spectra (νmax) were recorded on a Perkin–Elmer 1800
Series FTIR Spectrometer (Waltham, MA). Samples were applied to KBr plates as CDCl3
solutions followed by generous air drying. Lowꢀresolution ESI mass spectra were recorded
on a single quadrupole liquid chromatographꢀmass spectrometer, while highꢀresolution
measurements were conducted on a timeꢀofꢀflight instrument. Melting points were measured
on an Optimelt automated melting point system (Praxair Inc., Danbury, CT) and are
uncorrected. Analytical thin layer chromatography (TLC) was performed on aluminumꢀ
backed 0.2 mm thick silica gel 60 F254 plates as supplied by Merck (Darmstadt, Germany).
Visualization was accomplished with potassium permanganate solution. Flash
chromatographic separations were carried out with silica gel 60 (40–63 ꢀm) as the stationary
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phase and using the analytical reagent grade solvents indicated. Where necessary, reactions
were performed under an nitrogen atmosphere.
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Specific Chemical Transformations
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Methyl (1S,2S,4aR,4bR,7S,9aS,10S,10aR)-2,7-dihydroxy-1-methyl-8-methylene-13-oxo-
1,2,4b,5,6,7,8,9,10,10a-decahydro-4a,1-(epoxymethano)-7,9a-methanobenzo[a]azulene-
10-carboxylate (17). To a stirred solution of gibberellin A3 (3, 10.2 g, 29.38 mmol) in
acetone (800 mL) was added anhydrous potassium carbonate (8.93 g, 44.1 mmol) and methyl
iodide (11 mL, 176 mmol). The reaction mixture was stirred at 18 °C for 20 h. The resulting
suspension was filtered through CeliteTM, the filter cake rinsed with acetone (300 mL) and
concentrated under reduced pressure. The resulting white solid was subjected to
chromatography (silica, 2:3 v/v acetone/hexane elution) to afford, after concentration of the
relevant fractions (Rf = 0.1 in hexane/EtOAc 1:1), methyl gibberellate 17 (10 g, 94%) as a
white solid. The spectroscopic data was in good comparison with that previously described in
the literature.36 1H NMR (400 MHz, CDCl3) δ 6.32 (d, J = 9.2 Hz, 1H), 5.91 (dd, J = 9.2, 3.9
Hz, 1H), 5.28 (dd, J = 3.1, 1.9 Hz, 1H), 4.97 (d, J = 2.4 Hz, 1H), 4.16 (dd, J = 3.9, 0.9 Hz,
1H), 3.73 (s, 3H), 3.21 (d, J = 10.8 Hz, 1H), 2.79 (d, J = 10.8 Hz, 1H), 2.20 (m, 1H),
2.11−2.03 (complex m, 2H), 1.96−1.90 (complex m, 2H), 1.81−1.67 (complex m, 4H), 1.24
(s, 3H).
Methyl
(1S,2S,4aR,4bR,7S,9aS,10S,10aR)-1-methyl-8-methylene-2,7-
bis((methylsulfonyl)oxy)-13-oxo-1,2,4b,5,6,7,8,9,10,10a-decahydro-4a,1-
(epoxymethano)-7,9a-methanobenzo[a]azulene-10-carboxylate (18). To a magnetically
stirred mixture of methyl gibberellate 17 (10.0 g, 27.7 mmol) in pyridine (18 mL) was added,
dropwise, methanesulfonly chloride (5.37 mL, 69.4 mmol) at 0 °C. The ensuing yellow
solution was then stirred at 18 °C for 13 h before being poured into aqueous 1M HCl (200
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mL) and extracted with ethyl acetate (3 x 250 mL). The combined organic extracts were
washed with 1 M HCl (100 mL), brine (75 mL), then dried (Na2SO4), filtered and
concentrated under reduced pressure. The ensuing yellow residue was subjected to flash
chromatography (silica, 1:15 v/v ethyl acetate/dichloromethane elution) to afford, after
concentration of the relevant fractions (Rf = 0.7), dimesyl methyl gibberellate 18 (13.2 g,
92%) as an amorphous, white solid. The spectroscopic data was in good comparison with that
previously described in the literature.36 1H NMR (400 MHz, CDCl3) δ 6.46 (d, J = 9.2 Hz,
1H), 6.03 (dd, J = 9.2, 3.8 Hz, 1H), 538 (m, 1H), 5.15 (m, 1H), 5.07 (d, J = 3.8 Hz, 1H), 3.76
(s, 3H), 3.31 (d, J = 10.7 Hz, 1H), 3.10 (s, 3H), 3.04 (s, 3H), 2.81 (d, J = 10.7 Hz, 1H), 2.60
(m, 1H), 2.51−2.43 (complex m, 2H), 2.25−2.17 (complex m, 2H), 2.04−1.94 (complex m,
3H), 1.76 (m, 1H), 1.28 (s, 3H).
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(1R,4bR,7R,9aR,10S,10aS)-10-(methoxycarbonyl)-1-methyl-8-methylene-
2,3,4b,5,6,7,8,9,10,10a-decahydro-1H-7,9a-methanobenzo[a]azulene-1-carboxylic acid
(19). To a magnetically stirred mixture of dimesyl methyl gibberellate 18 (3.31 g, 6.41 mmol)
in dry THF (26 mL), tertꢀbutyl alcohol (2.7 mL, 37.8 mmol) and liquid ammonia (100 mL,
distilled with a piece of lithium) at –78 °C was added lithium metal (289 mg, 41.7 mmol) in
small pieces over a period of 5 hours. The reaction was monitored via TLC by taking small
aliquots of the reaction mixture and quenching with 1M HCl and diluting with ethyl acetate.
Upon reaction completion, solid NH4Cl (10 g) was added and the ammonia was left to
evaporate overnight. The resulting residue was brought to pH 4 with 1M HCl and extracted
with ethyl acetate (3 × 100 mL). The combined organic extracts were washed with 1M
NaH2PO4 solution (100 mL), dried (Na2SO4), filtered and concentrated under reduced
pressure. The ensuing yellow residue obtained from four parallel reactions were combined
and subjected to flash chromatography (silica, 1:9:0.1 v/v/v ethyl acetate/hexane/AcOH
elution) to afford, after concentration of the relevant fractions (Rf = 0.8), acid 19 (5.0 g, 59%)
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as an amorphous, white solid. The spectroscopic data was in good comparison with that
previously described in the literature.21 1H NMR (400 MHz, CDCl3) δ 5.35 (m, 1H), 4.87
(broad s, 2H), 3.70 (s, 3H), 3.03 (d, J = 5.5 Hz, 1H), 2.88 (m, 1H), 2.55−2.50 (m, 2H),
2.43−2.38 (m, 2H), 2.22−2.08 (complex m, 4H), 1.76−1.61 (complex m, 2H), 1.58−1.43
(complex m, 4H), 1.24 (s, 3H), 1.20 (m, 2H).
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Methyl
(1S,1aR,4R,4aS,5S,5aR,8R,10aR,10bR)-4-methyl-7-methylene-12-
oxododecahydro-1H-1,4:5a,8-dimethanocyclopropa[1,6]benzo[1,2-a]azulene-5-
carboxylate (23). Step i: To a magnetically stirred mixture of oxalyl chloride (10.2 mL,
120.8 mmol) in dry Et2O (60 mL) at 0 °C was added, dropwise a solution of acid 19 (5.0 g,
15.1 mmol) in dry Et2O (80 mL) and pyridine (20 mL). Upon completion of addition, the
reaction was allowed to warm to 18 °C, dry DMF (1 mL) was added and the reaction was left
to stir for 24 h. The resulting suspension was filtered through CeliteTM under a stream of
nitrogen and the filter cake was rinsed thoroughly with Et2O (6 × 100 mL). The combined
filtrates were concentrated under reduced pressure and the higher boiling point components
azeotrophed with dry toluene (100 mL) to provide the crude acyl chloride 20 as a yellow oil,
which was subjected to next step directly. Step ii: The crude acyl chloride 20 obtained was
dissolved in dry Et2O (50 mL) and added, dropwise, to a stirred solution of diazomethane in
ether (170 mL of a 0.9 M solution in diethyl ether, prepared from the known literature
procudure37) at 0 °C. Upon completion of addition the solution was allowed to warm to 18 °C
over 16 h. Careful evaporation of the excess diazomethane and removal of Et2O provided the
crude diazoketone 21 as a yellow oil, which was subjected to next step directly. Step iii: The
obtained crude diazoketone 21 was dissolved in dry THF and cyclohexane (80 mL, 1:1 v/v)
and added, dropwise to a refluxing solution of bis(Nꢀtertꢀbutylsalicylaldiminato)copper(II) 22
(586 mg, 1.4 mmol) in cyclohexane (100 mL). The ensuing dark red solution was heated at
reflux 2 h before being allowed to cool to room temperature and concentrated under reduced
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pressure. The dark red residue obtained was diluted with Et2O (200 mL) and washed with
10% aqueous solution of ammonia (200 mL), the aqueous layer was further extracted by Et2O
(3 × 100 mL), and the combined organic extracts washed with 1M NaH2PO4 (100 mL), brine
(100 mL), dried with anhydrous NaSO4, filtered and concentrated under reduced pressure.
The ensuing residue was subjected to flash chromatography (silica, 2:98 v/v ethyl
acetate/dichloromethane elution) to afford, after concentration of the relevant fractions (Rf =
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20
0.5), cyclopropane 23 (2.6 g, 53% over 3 steps) as a colourless oil, [α]D = −90.4 (c 1,
1
CHCl3). H NMR (400 MHz, CDCl3) δ 4.94 (broad s, 1H), 4.87 (broad s, 1H), 3.68 (s, 3H),
2.70 (d, J = 10.4 Hz, 1H), 2.59 (t, J = 6.4 Hz, 1H), 2.36 (d, J = 10.4 Hz, 1H), 2.22−2.16
(complex m, 2H), 2.03−1.96 (complex m, 2H),1.95−1.87 (complex m, 2H), 1.82 (d, J = 8.2
Hz, 1H), 1.76−1.72 (complex m, 1H), 1.67−1.61 (complex m, 2H), 1.60−1.52 (complex m,
2H), 1.39−1.29 (complex m, 1H), 1.27−1.17 (complex m, 1H), 0.91 (complex m, 1H), 0.82
(s, 3H). 13C NMR (100 MHz, CDCl3) δ 215.0, 173.9, 156.9, 107.3, 53.0, 52.1, 51.7, 50.0,
47.8, 46.5, 46.3, 44.1, 38.9, 37.8, 36.9, 31.8, 31.5, 30.9, 18.9, 17.6, 15.5. IR (KBr): νmax 2933,
2863, 1723, 1658, 1435, 1375, 1260, 1191, 1165, 1029, 991, 881 cmꢀ1. MS (ESI, +ve): m/z
23
349 ([M+Na]+, 100%). HRMS [M+Na]+ Calcd for C21H26O3 Na: 349.1780. Found:
349.1780.
Methyl (1R,4aR,4bS,7R,9aR,10S,10aS)-1-methyl-8-methylene-13-oxododecahydro-1,4a-
ethano-7,9a-methanobenzo[a]azulene-10-carboxylate (24). Step i: To a magnetically
stirred mixture of cyclopropane 23 (2.6 g, 8.0 mmol) in dry THF (100 mL), tertꢀbutyl alcohol
(2.2 mL, 24.0 mmol) and liquid ammonia (200 mL, distilled with a piece of lithium) at −78
°C was added lithium metal (0.14 g, 20.0 mmol) in small pieces over a period of 5 hours. The
reaction was monitored via TLC by taking small aliquots of the reaction mixture and
quenching with 1M HCl and diluting with ethyl acetate. Upon reaction completion, solid
NH4Cl (10 g) was added and the ammonia was left to evaporate overnight. The resulting
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residue was brought to pH 4 with 1M HCl and extracted with ethyl acetate (3 × 100 mL).
The combined organic extracts were washed with 1M NaH2PO4 solution (100 mL), dried
(Na2SO4), filtered and concentrated under reduced pressure. The ensuing yellow residue was
subjected to next step directly. Step ii: A magnetically stirred mixture of the aboveꢀmentioned
crude material in dry dichloromethane (50 mL) and pyridine (0.27 mL, 3.4 mmol) at 0 °C
was treated with DessꢀMartin Periodinane (1.7 g, 4.0 mmol) and the reaction mixture was left
to stir for 5 h. Removal of the solvent and dilution with diethyl ether produced a white
precipitate which was filtered through CeliteTM, the filter cake was rinsed with additional
diethyl ether (50 mL× 3), and the filtrate was concentrated under reduced pressure to afford a
yellow oil. Of which was then subjected to flash chromatography (silica, 2:98 v/v ethyl
acetate/dichloromethane elution) to afford, after concentration of the relevant fractions (Rf =
0.5), ketone 24 (2.0 g, 77% over 2 steps) as an amorphous, white powder, [α]D20 = −30.0 (c
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1
1.1, CHCl3). H NMR (400 MHz, CDCl3) δ 4.95 (broad s, 1H), 4.83 (broad s, 1H), 3.67 (s,
3H), 2.62 (m, 1H), 2.42 (d, J = 11.9 Hz, 1H), 2.34 (d, J = 11.9 Hz, 1H), 2.16−2.02 (complex
m, 3H), 1.94−1.89 (complex m, 1H), 1.86−1.78 (complex m, 2H), 1.71−1.62 (complex m,
3H), 1.58−1.49 (complex m, 4H), 1.48−1.33 (complex m, 3H), 1.21−1.12 (complex m, 1H),
0.90 (s, 3H).13C NMR (100 MHz, CDCl3) δ 220.8, 173.8, 157.6, 106.7, 59.0, 54.8, 53.5, 52.4,
51.6, 51.0, 49.2, 45.4, 43.7, 39.0, 38.1, 36.6, 36.3, 31.8, 19.9, 19.0, 17.1. IR (KBr): νmax 2933,
2867, 1736, 1656, 1439, 1375, 1285, 1194, 1171, 1087, 1016, 878 cmꢀ1. MS (ESI, +ve): m/z
23
351 ([M+Na]+, 19%), 301 (100). HRMS [M+Na]+ Calcd for C21H28O3 Na: 351.1936. Found:
351.1937.
(1R,4aR,4bR,7R,9aR,10S,10aS)-4a-Formyl-10-(methoxycarbonyl)-1-methyl-8-
methylenedodecahydro-1H-7,9a-methanobenzo[a]azulene-1-carboxylic acid (25). A
magnetically stirred mixture of ketone 24 (2.0 g, 6.1 mmol) in tetrahydrofuran (50 mL) and
dimethylformamide (50 mL) at 0 °C was treated with potassium hexamethyldisilazide (14.6
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mL of a 0.5 M solution in toluene, 7.3 mmol). The mixture was stirred at 0 °C for 1 h before
being bubbled with oxygen gas. After a further 2 h, the reaction was quenched with water (1
mL). The solvent was removed under reduced pressure and the resulting solid mixed with a
small amount of silica, dissolved in dichloromethane and dried to a fine powder which was
subjected to flash chromatography (silica, 1:4:0.05 v/v/v ethyl acetate/hexane/acetic acid
elution) to afford, after concentration of the relevant fractions (Rf = 0.3), lactol 25 (1.7 g,
77%) as a white crystalline solid (m.p. 165−168 °C), [α]D20 = −76.0 (c 0.5, CHCl3). This
compound is not suitable for NMR analysis due to lactol resonances. IR (KBr): νmax 3377,
2940, 2877, 1732, 1436, 1381, 1266, 1237, 1195, 1169, 1123, 983, 916, 807 cmꢀ1. MS (ESI,
+ve): m/z 383 ([M+Na]+, 23%), 744 ([2M+Na]+, 100). HRMS [M+Na]+ Calcd for
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C21H28O5 Na: 383.1834. Found: 383.1834.
Further structural determination of lactol 25 was achieved on the corresponding methyl
ester, which was prepared by treating compound 25 (10 mg) with a solution of diazomethane
in ether until the yellow colour persisted. The solution was concentrated under reduced
1
pressure. The resulting methyl ester 26 was then submitted to structural analysis. H NMR
(400 MHz, CDCl3) δ 9.65 (s, 1H), 4.90 (broad s, 1H), 4.82 (broad s, 1H), 3.86 (d, J = 12.6
Hz, 1H), 3.70 (s, 3H), 3.62 (s, 3H), 2.60 (t, J = 6.1 Hz, 1H), 2.37 (dd, J = 13.0, 3.4 Hz, 1H),
2.22 (d, J = 12.6 Hz, 1H), 2.18−2.09 (complex m, 2H), 1.98 (dt, J = 15.9, 2.9 Hz, 1H), 1.83
(m, 1H), 1.69−1.61 (complex m, 1H), 1.62−1.58 (complex m, 2H), 1.57−1.50 (complex m,
3H), 1.40−1.31 (complex m, 1H), 1.25−1.15 (complex m, 1H), 1.12 (s, 3H), 1.11−1.08
(complex m, 1H), 0.95 (td, J = 13.0, 4.5 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 205.5,
176.6, 175.1, 155.8, 106.5, 60.4, 56.6, 56.0, 51.6, 51.6, 50.3, 50.0, 45.8, 45.2, 39.5, 38.3,
37.8, 33.0, 31.9, 28.1, 20.8, 17.7. IR (KBr): νmax 2939, 2871, 1733, 1435, 1376, 1262, 1231,
1196, 1169, 1168, 1147 cmꢀ1. MS (ESI, +ve): m/z 397 ([M+Na]+, 100%), 375 ([M+H]+, 33).
23
HRMS [M+Na]+ Calcd for C22H30O5 Na: 397.1991. Found: 397.1996.
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GA24 (10). To a magnetically stirred mixture of lactol 25 (1.7 g, 4.72 mmol) in methanol (12
mL) was added NaOH aqueous solution (2M, 60 mL) and the mixture was heated at 100 °C
for 20 h. The volatile solvent was then removed, the pH was adjusted to 4 with 1 M HCl, and
the aqueous layer was extracted with ethyl acetate (3 × 75 mL). The combined organic
extracts were dried (Na2SO4), filtered and concentrated under reduced pressure. The ensuing
residue was subjected to flash chromatography (silica, 1:2:0.05 v/v/v ethyl
acetate/hexane/acetic acid elution) to afford, after concentration of the relevant fractions (Rf =
0.45), GA24 10 (1.5 g, 87%) as a white, crystalline solid, m.p. 203−205 °C (MeOH/EtOAc)
{lit38. 198−203 °C}, [α]D20 = −105.0 (c 0.8, EtOH) {lit38. −88 (c 0.6, EtOH)}.GA24 is not
suitable for NMR analysis due to lactol resonances. IR (KBr): νmax 3336, 2939, 2875, 1705,
1407, 1287, 1267, 1194, 1147, 1126, 977, 950, 876 cmꢀ1. MS (ESI, +eV): m/z 369 ([M+Na]+,
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95%), 715 ([2M+Na]+, 100). HRMS [M+Na]+ Calcd for C20H26O5 Na: 369.1678. Found:
369.1678.
Further structural determination of GA24 was achieved on the corresponding diꢀmethyl
ester 26, which was prepared by treating GA24 (10 mg) with a solution of diazomethane in
ether until the yellow colour persisted. The solution was concentrated under reduced pressure.
The ensuing diꢀmethyl ester showed the same structural data, in all respects, to compound 26
obtained previously.
GA15 (9). To a magnetically stirred mixture of GA24 10 (1.0 g, 2.9 mmol) in ethanol (20 mL)
at −10 °C was added, portionꢀwise, sodium borohydride (378 mg, 10.0 mmol). The mixture
was stirred at −10 °C for 5 h. The excess sodium borohydride was quenched with acetone (3
mL) followed by 1M HCl (20 mL), and the mixture was left to stir for 5 min before the
volatile solvent was removed under reduced pressure. The solution was then extracted with
ethyl acetate (3 × 75 mL), the combined organic extracts were dried (Na2SO4), filtered and
concentrated under reduced pressure. The ensuing residue was subjected to flash
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chromatography (silica, 1:3:0.05 v/v/v ethyl acetate/hexane/acetic acid elution) to afford,
after concentration of the relevant fractions (Rf = 0.8), GA15 9 (0.87 g, 92%) as a white,
crystalline solid, m.p. 266−270 °C (MeOH/EtOAc) {lit39 274−276 °C}, [α]D20 = −83.6 (c
1
0.55, CHCl3) {lit39 +5.0 (no concentration and solvent indicated)}. H NMR (400 MHz,
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CDCl3) δ 4.94 (broad s, 1H), 4.82 (broad s, 1H), 4.40 (d, J = 12.1 Hz, 1H), 4.10 (d, J = 12.1
Hz, 1H), 2.79 (d, J = 12.7 Hz, 1H), 2.65 (m, 1H), 2.19−2.10 (complex m, 2H), 2.08−1.99
(complex m, 3H), 1.86−1.82 (complex m, 1H), 1.75−1.71 (complex m, 2H), 1.64−1.54
(complex m, 3H), 1.51−1.41 (complex m, 2H), 1.40−1.31 (complex m, 2H), 1.20 (s, 3H),
1.11−1.02 (complex m, 1H). 13C NMR (100 MHz, CDCl3) δ178.2, 175.3, 156.4, 106.9, 74.4,
55.9, 52.7, 52.0, 49.8, 46.1, 42.8, 41.9, 40.0, 39.4, 38.4, 36.5, 31.4, 23.3, 20.8, 16.0. IR
(KBr): νmax 3196, 2935, 2872, 1724, 1422, 1262, 1194, 1156, 1036, 912, 879, 732 cmꢀ1. MS
(ESI, +ve): m/z 353 ([M+Na]+, 21%), 683 ([2M+Na]+, 100). HRMS [M+Na]+ Calcd for
23
C20H26O4 Na: 353.1729. Found: 353.1728.
ASSOCIATED CONTENT
Supporting Information
13
1H NMR spectra of compounds 17ꢀ19, 1H and C NMR spectra of compounds 9, 23, 24 and
26. This material is available free of charge via the internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Telephone: +86ꢀ20ꢀ85221367. Fax: +86ꢀ20ꢀ85224766.
Eꢀmail: lew.mander@anu.edu.au. (L. N. M.)
Eꢀmail: ping.lan@jnu.edu.cn. (P. L.)
ORCID
David J.ꢀY. D. Bon: 0000ꢀ0001ꢀ7867ꢀ6877
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Ping Lan: 0000ꢀ0002ꢀ9285ꢀ3259
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Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
The authors thank the Fundamental Research Funds for the Central Universities (grant
21617327), China and the Chinese National Natural Science Foundation (grant 31700670),
the Guangdong Natural Science Foundation (grant 2017A030313202), as well as the
Department of Science and Technology of Guangdong Province under grant
2016A010105010 for financial support.
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