Synthesis and biological activity of tetralone abscisic acid analogues

Article abstract of DOI:10.1039/b509193d

Bicyclic analogues of the plant hormone abscisic acid (ABA) were designed to incorporate the structural elements and functional groups of the parent molecule that are required for biological activity. The resulting tetralone analogues were predicted to have enhanced biological activity in plants, in part because oxidized products would not cyclize to forms corresponding to the inactive catabolite phaseic acid. The tetralone analogues were synthesized in seven steps from 1-tetralone and a range of analogues were accessible through a second route starting with 2-methyl-1-naphthol. Tetralone ABA 8 was found to have greater activity than ABA in two bioassays. The absolute configuration of (+)-8 was established by X-ray crystallography of a RAMP hydrazone derivative. The hydroxymethyl compounds 10 and 11, analogues for studying the roles of 8′- and 9′-hydroxy ABA 3 and 6, were also synthesized and found to be active. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b509193d

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Synthesis and biological activity of tetralone abscisic acid analogues†
James M. Nyangulu,‡a Ken M. Nelson,a Patricia A. Rose,a Yuanzhu Gai,a Mary Loewen,a Brenda Lougheed,a
J. Wilson Quail,b Adrian J. Cutlera and Suzanne R. Abrams*a
Received 29th June 2005, Accepted 3rd February 2006
First published as an Advance Article on the web 1st March 2006
DOI: 10.1039/b509193d
Bicyclic analogues of the plant hormone abscisic acid (ABA) were designed to incorporate the
structural elements and functional groups of the parent molecule that are required for biological
activity. The resulting tetralone analogues were predicted to have enhanced biological activity in plants,
in part because oxidized products would not cyclize to forms corresponding to the inactive catabolite
phaseic acid. The tetralone analogues were synthesized in seven steps from 1-tetralone and a range of
analogues were accessible through a second route starting with 2-methyl-1-naphthol. Tetralone ABA 8
was found to have greater activity than ABA in two bioassays. The absolute configuration of (+)-8 was
established by X-ray crystallography of a RAMP hydrazone derivative. The hydroxymethyl compounds
10 and 11, analogues for studying the roles of 8ꢀ- and 9ꢀ-hydroxy ABA 3 and 6, were also synthesized
and found to be active.
Introduction
The plant hormone abscisic acid (ABA, 1, Scheme 1) regulates
many aspects of plant growth and development as well as responses
to environmental stress.1 For example, in seed development ABA
induces synthesis of storage products, prevents germination of
immature embryos and is involved in desiccation tolerance and
germination of mature seed.1,2 ABA levels in plants increase
transiently in response to environmental stress and trigger a set
of rapid responses including closure of the stomata, reducing
transpiration.
Numerous studies have been conducted to probe the structural
requirements of ABA responses to develop analogues that are
effective plant growth regulators.3–7 Some features of the ABA
molecule appear to be required for activity, particularly the
carboxyl and ketone groups, the six-membered ring, the 7ꢀ-methyl
group, and the 2-Z configuration of the double bond in the side
chain. Other parts of the molecule can be modified without loss of
activity. The ring double bond, both the 8ꢀ- and 9ꢀ-methyl groups,
and the 4-E double bond of the side chain each can be altered and
the resultant analogue retains activity.
Scheme 1 Metabolism pathways of ABA (1).
group affording 8ꢀ-hydroxy ABA 3 which is in equilibrium with
the closed form phaseic acid 4.9 Alternative pathways, through
hydroxylation of the 7ꢀ-methyl group affording 7ꢀ-hydroxy ABA 5
and the 9ꢀ-methyl group to give 9ꢀ-hydroxy ABA 6, which can also
rearrange to the closed form neo-phaseic acid 7, have also been
observed and contribute to ABA catabolism.10,11 Rapid catabolism
by plant enzymes limits the practical application of ABA itself as a
plant growth regulator.7 Metabolism resistant analogues of ABA
altered at the 8ꢀ carbon atom have proved to be more persistent
and more active than ABA.7
We considered that the tetralone ABA analogue 8, (Fig. 1) in
which the planar vinyl methyl portion of ABA has been replaced
with an aromatic ring, had potential to have strong biological
activity as it would be unable cyclize to forms corresponding to
the inactive catabolite phaseic acid 4. The essential features of the
ABA molecule are maintained, with preservation of the ABA side
chain, the C-4ꢀ ketone group, the stereochemistry at C-1ꢀ and the
These structure–activity results are also important for devel-
oping analogues to assess the bioactivity of metabolites, and to
produce probes for identifying ABA binding proteins. As shown
in Scheme 1, ABA is catabolized predominantly through oxidation
of the ring methyl groups or alternatively by conjugation to the
glucose ester 2.7–11 The principal pathway of oxidation is through
P450 monooxygenase-mediated hydroxylation of the 8ꢀ-methyl
aPlant Biotechnology Institute, National Research Council of Canada,
110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9. E-mail:
sue.abrams@nrc-cnrc.gc.ca; Fax: 306 975 4839; Tel: 306 975 5333
bDepartment of Chemistry and Saskatchewan Structural Sciences Centre,
University of Saskatchewan, 110 Science Place, Saskatoon, SK, Canada
S7N 5C9
† Dedicated to the memory of Angela C. Shaw.
‡ Current address: Department of Chemistry, St Francis Xavier University,
PO Box 5000, Antigonish, NS, Canada B2G 2W5. Fax: 902 867 2196; Tel:
902 867 5259; Email: jnyangul@stfx.ca
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Fig. 1 ABA analogues.
planar geometry of C-3ꢀ and C-7ꢀ. We predicted that the tetralone
ABA analogue 8 would have activity as it had been shown in a
previous study that a related tricyclic analogue ABA 9 had weak
ABA-like response in a growth inhibition assay.12
Scheme 2 (a) CH3I, NaH, THF; (b) 14, THF; (c) RedAl, THF; (d) MnO2,
acetone; (e) MnO2, NaCN, HOAc, MeOH; (f) PDC, tert-BuOOH, PhH;
(g) chiral HPLC; (h) KOH, MeOH(aq).
Tetralone ABA 8 and related hydroxylated compounds 10 and
11 are novel ABA analogues for probing the biological activity
of ABA and its labile catabolites. We considered that a P450
monooxygenase in corn cells that hydroxylates ABA might accept
the tetralone ABA analogue 8 as a substrate and generate a
hydroxymethyl compound 10 (analogous to 8ꢀ-hydroxy ABA 3).
It was also predicted that the hydroxymethyl derivative, would not
cyclize to a phaseic acid- like compound, as conjugate addition of
the hydroxyl oxygen to the enone would be prevented, preserving
the aromaticity of the fused ring, and that 10 could be employed
in bioassays as a robust analogue to probe the role of 8ꢀ-hydroxy
ABA. Similarly 11, the analogue of 9ꢀ-hydroxy ABA, would be
useful for probing the activity of the natural catabolite 6.
oxidation of 17 using a combination of pyridinium dichromate
and tert-butyl hydroperoxide yielded racemic methyl ester 18.16
The ester 18 was resolved by preparative HPLC using a column
with a chiral ligand. Base hydrolysis of the enantiomers yielded
the respective enantiopure (+)- and (−)-isomers of tetralone ABA
acid 8. The absolute stereochemistry was assigned from X-ray
crystallographic analysis of a derivative of the (+)-enantiomer
(described below).
Conformational mobility of tetralone analogues
Here we report syntheses of tetralone ABA analogues 8, 10 and
11. An alternate synthetic route for the production of tetralone
ABA analogues with a wide range of 9ꢀ-substituents (analogous
to 8ꢀ-substituted ABAs) is also discussed. The biological activity of
the tetralone ABA analogue 8 is compared to that of ABA in two
assays and found to be more potent. The tetralone ABA analogue
can be used to probe biological activity of ABA catabolites and
for affinity probes for identifying ABA binding proteins.13 The
bioactivity of hydroxylated compounds 10 and 11 is also reported.
The 1H NMR spectrum of the methyl ester 18 shows broadening
of peaks, especially the H-4 (d 7.82 ppm) of the side chain as well
as the a-methylene protons at C-3ꢀ of the ring (d 2.5–2.9 ppm),
typically a sign of restricted rotation around C-1ꢀ. This phe-
nomenon had been observed previously with a C-1ꢀ methyl ether
ABA analogue.5 Through variable temperature 1H NMR, the peak
broadening for the C-1ꢀ methyl ether ABA had been attributed
to the barrier to interconversion between conformations with
sidechain–axial and sidechain–equatorial.
Results and discussion
Absolute configuration of analogue (+)-8
Synthesis of tetralone ABA analogue 8
The absolute stereochemistry at C-1ꢀ of the tetralone analogue
(+)-8 was established by X-ray crystallography of a tetralone
ABA derivative, which was synthesized from the tetralone ABA
18 (Scheme 3). The condensation of racemic 18 with commercially
available (R)-1-amino-2-methoxymethyl-pyrrolidine (RAMP) 19
in the presence of para-toluenesulfonic acid (PTSA) gave a mixture
of two diastereomers of the hydrazone 20,17 which were separable
by column chromatography. One of the diastereomers was charac-
terized as follows: reduction of the ester group, followed by allylic
oxidation of the resulting alcohol 21, afforded the aldehyde 22.
Condensation of the aldehyde 22 with dansyl hydrazine in the
presence of trichloroacetic acid gave the derivative 23, which gave
crystals suitable for X-ray analysis. As the absolute configuration
of one of the stereogenic centers (2ꢀꢀR) was known, the absolute
Racemic tetralone methyl abscisate 18 was synthesized from
commercially available 1-tetralone 12 (Scheme 2). The geminal
methyl groups were introduced adjacent to the carbonyl carbon
by treatment of 1-tetralone with methyl iodide in the presence of
sodium hydride to give the dimethyl tetralone 13 in 83% yield.14
The side chain was introduced by standard methods.15 Alkylation
of the dimethyl tetralone 13 with dilithium salt 14, gave the key
intermediate 15 in 64% yield. Reduction of the triple bond using
R
RedAlꢀ yielded the allylic alcohol 16. Successive oxidations with
manganese dioxide to the aldehyde, confirmed by the appearance
of an aldehyde doublet in crude 1H NMR and then with a
combination of manganese dioxide, sodium cyanide, acetic acid in
methanol, gave the ester 17 in 19% yield over three steps. Benzylic
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Scheme 3 (a) PTSA; (b) LiAlH4; (c) MnO2; (d) CCl3CO2H–dansyl
hydrazine; (e) oxalic acid.
Scheme 4 (a) PhI(OAc)2–ethylene glycol; (b) 14, THF; (c) RedAl;
(d) MnO2; (e) MnO2–NaCN–AcOH–MeOH; (f) 10% HCl (aq); (g)
HC2MgBr; (h) chiral HPLC; (i) KOH, MeOH(aq).
configuration at C-1ꢀ was determined to be (S), as shown from the
crystal structure (Fig. 2). The hydrazone 20 was hydrolyzed in the
presence of oxalic acid to afford (+)-18, as determined by HPLC
using the chiral column. The observed biological assay results
for the tetralone analogue (+)-8 are consistent with the biological
activity in similar assays observed for natural (+)-ABA which also
has the C-1ꢀ (S) absolute stereochemistry.18
analogues resistant to metabolism and the putative metabolite
of (+)-8. More persistent ABA analogues with 8ꢀ-methylene and
8ꢀ-acetylene substituents have been synthesized and shown to
have strong biological activities.6 For example, with commercially
available 2-methyl-1-naphthol (24) as starting material, the methyl
substituted ketal 25, was obtained through oxidation using
iodobenzene diacetate. Alkylation with dilithium salt 14, followed
by triple bond reduction to intermediate 27, two successive
oxidations and deprotection of ketal 28 leads to the enone 29.
The 9ꢀ-acetylene group was introduced by the conjugate addition
of ethynylmagnesium bromide to the enone 29 to afford methyl
9ꢀ-acetylene tetralone ABA 30. Resolution of 30 by chiral HPLC
followed by base hydrolysis gave enantiomers (+)-31 and (−)-31. It
has previously been shown that such conjugate additions afforded
the product with the alkyl group adding to the same face of the
molecule as the hydroxyl group at C-1ꢀ.6,19 As well, irradiation
1
of the 10ꢀ-methyl group of 30 (1.41 ppm) in the H NMR gave
a positive NOE of the proton on the side chain (H-5 doublet at
7.70 ppm), which is consistent with structure 30.
Metabolism of (+)-8
We conducted metabolism studies to determine if the bicyclic
ABA analogue 8 would be a substrate of P450 monooxygenases
in corn suspension-cultured cells. Metabolism studies have been
conducted in maize (Zea mays L. CV. Black Mexican Sweet)
suspension-cultured cells, which had previously been shown to
rapidly metabolize (+)-ABA to (−)-PA by hydroxylation of the
8ꢀ-methyl group.20 The substrate ABA and metabolites were found
to accumulate in the medium of the cell suspension culture. We
therefore anticipated that the enantiomer of 8 like (+)-ABA
would be converted to the hydroxylated compound analogous
Fig. 2 Crystal structure of compound 23.
Synthesis of methyl modified tetralone ABA analogues
An alternate synthetic route (Scheme 4) was developed to produce
tetralone analogues that could have a wide range of substituents
at the C-9ꢀ (8ꢀ-substituted following ABA numbering), such as
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to 8ꢀ-hydroxyABA. We also predicted that the enantiomer of
8 similar to (−)-ABA would not be converted to a compound
analogous to (−)-7ꢀ-hydroxyABA, the metabolite of (−)-ABA due
to the presence of the aromatic ring in compound 8.6 Suspension-
cultured corn cells were incubated with the (+) and the (−)-
enantiomers of tetralone ABA analogue 8 for 4 d (Fig. 3). The
(+)-isomer was rapidly consumed (only 20% remained after 45 h),
at a rate similar to that observed for (+)-ABA.21 A single
metabolite was isolated from the culture medium. Proton NMR
of the isolated metabolite showed the disappearance of the 9ꢀ-
methyl group at d 1.05 ppm and the appearance of a doublet
centered at d 3.65 ppm. On the basis of the known mode of
action of P450 monooxygenase in corn cells and other spectral
1
data (mass, H NMR, optical rotation and IR), the metabolite
was confirmed to be the bicyclic 9ꢀ-hydroxy tetralone ABA (+)-
10. The P450 monooxygenase in corn cells that metabolize ABA
to 8ꢀ-hydroxyABA is likely also responsible for hydroxylation of
the bicyclic ABA analogue. The analogue (+)-10 accumulates in
the corn cell medium (Fig. 3), unlike 8ꢀ-hydroxyABA 2, which in
corn cell culture, the equilibrium favors PA 4.21
Scheme 5 (a) NaOCH3–ethyl formate; (b) CH3I–KOH; (c) MeOH–
NH4Cl; (d) 14, THF; (e) RedAl; (f) MnO2; (g) MnO2–NaCN–AcOH–
MeOH; (h) PDC–tert-BuOOH; (i) 50% TFA–CHCl3; (j) NaBH4;
(k) MnO2; (l) chiral HPLC; (m) KOH, MeOH(aq).
Fig. 3 Metabolism in maize suspension culture of enantiomer (+)-(S)-8.
two diastereomeric allylic alcohols (precursors for the 9ꢀ- and 10ꢀ-
hydroxylated ABA analogues) from the same intermediate 34 after
side chain alkylation.
The concentration of the (−)-enantiomer of the bicyclic ana-
logue 8, under the same biotransformation conditions slowly
decreased so that at 96 h, 50% remained and no hydroxylated
product was observed. In previous studies (−)-ABA was found to
be slowly transformed principally to (−)-7ꢀ-hydroxy ABA.21
Although these studies were performed with intact corn cells
with multiple P450 monooxygenases, we are able to demonstrate
that the monooxygenases present can accommodate the steric bulk
of the bicyclic analogue (+)-8.
Acetal 34 was obtained from readily available 1-tetralone 12, by
first introducing the hydroxymethylene group at the a-position of
1-tetralone using ethyl formate and sodium methoxide to give the
hydroxymethylene tetralone 32.22 Methylation of 32 was achieved
using methyl iodide in the presence of potassium hydroxide,
which gave the required aldehyde 33 as well as a by-product
formed by direct methylation of the hydroxyl group of 32, which
could be de-methylated and re-used to produce more of the
required aldehyde 33.23 The dimethyl acetal 34 was obtained by
refluxing 33 in methanol in the presence of a catalytic amount
of ammonium chloride, in quantitative yield.24 Alkylation of the
keto dimethyl acetal 34 with dilithium salt 14 gave a mixture of two
diastereomeric allylic alcohols, 35 and 36, in 1.7 : 1 ratio, which
were readily separable by column chromatography.
Encouraged by these results, we undertook to synthesize bicyclic
9ꢀ- and 10ꢀ-hydroxylated analogues to be used for probing the
biological activities of the more labile 8ꢀ- and 9ꢀ-hydroxy ABA in
intact plants.
Synthesis of 9ꢀ- and 10ꢀ-hydroxytetralone ABA analogues
A number of routes were attempted for the synthesis of the bicyclic
9ꢀ- and 10ꢀ-hydroxylated ABA analogues. The most efficient in
our hands had as a key intermediate, the protected aldehyde
34 (Scheme 5). The major advantage of this synthetic route is
that having the racemic mixture of 34, allows us to produce the
R
RedAlꢀ reduction of the triple bond in 35 and successive
oxidations of the resulting allylic alcohol with manganese oxide
to the aldehyde, and then with manganese oxide in the presence
of sodium cyanide and acetic acid affording the corresponding
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aldehyde, gave the ester 37 in 37% yield. Benzylic oxidation of the
ester 38, followed by de-acetalisation with 50% aqueous TFA in
chloroform mixture yielded the aldehyde 39.16 Reduction of the
aldehyde 39 with sodium borohydride and subsequent oxidation
of the crude trihydroxy ester with manganese oxide gave the
dihydroxy ester 40, which was resolved on a chiral HPLC column
to afford the corresponding (+)-40 and (−)-40 enantiomers. Base
hydrolysis of the respective enantiomers of 40 using KOH in
methanol gave the corresponding enantiomeric bicyclic 9ꢀ-hydroxy
ABA acids (61% yield) whose 1H NMR spectra were identical to
that of (+)-10 obtained from metabolism studies of (+)-8 (vide
supra).
The allylic alcohol 36 was treated the same way as compound
35, to give the corresponding enantiomeric 10ꢀ-hydroxytetralone
ABA acids (+)-11 and (−)-11.
Biological activity studies of tetralone ABA analogues
Fig. 5 Maize cell growth inhibition assay for (+)-ABA 1, (+)-10, and
(+)-11.
Growth inhibition assays. The growth inhibition assay using
suspension-cultured corn cells is a well characterized experimental
system that has been very useful for comparing the biological
activity and metabolism of ABA and ABA analogues.9 In this
study, the tetralone ABA analogue (+)-8, like (+)-ABA, inhibited
the growth of suspension-cultured cells of maize (Black Mexican
Sweet) in a dose-dependent manner over a concentration range
of 0.1–10.0 lM. As shown in Fig. 4, the analogue (−)-8 showed
inhibitory activity that is significantly higher than that of (+)-
ABA. Both enantiomers of 9ꢀ-acetylene tetralone ABA 31 also
had higher activity than ABA (+)-1 in this assay.
11 were comparable at 1 and 10 lM, but (+)-10 was apparently
less effective at 10 lM than (+)-11.
Arabidopsis seed germination assays. The tetralone ABA (+)-
8 was also studied in a germination assay of Arabidopsis thaliana
(Columbia wild type) seeds over a range of concentrations (0.33–
33 lM) (Fig. 6). Similar treatments were performed for ABA
1 (both enantiomers) to allow for a direct comparison between
ABA and the tetralone ABA 8. The results are expressed in
terms of germination indices, which summarize the rate and
extent of germination over the time of the experiment at a given
concentration. As shown in Fig. 6, the (+)-enantiomer of tetralone
ABA 8 was highly effective in inhibiting the germination of
the seeds over the 7-day test period at all concentrations. The
(+)-enantiomer of 8 is a more effective germination inhibitor
than (+)-ABA. At the lowest concentration of 0.33 lM, the
germination index for (+)-ABA (1) was almost 0.4, compared
with less than 0.1 in the case of (+)-tetralone ABA 8. As expected,
the (−)-enantiomer of the tetralone ABA analogue 8 was less
effective than the corresponding (+)-enantiomer. It was only active
at concentrations of ≥1 lM. A similar pattern was observed
Fig. 4 Maize cell growth inhibition assay for (+)-ABA 1, (+)-8, (+)-31
and (−)-31.
As observed in other studies with the maize suspension culture,
low concentrations of 1 were found to promote and higher
concentrations inhibit growth (Fig. 5).3 Concentrations of 1.0
micromolar or greater of (+)-8 and both (+)- and (−)-31
showed significant stronger inhibitory activity than (+)-1. In a
separate experiment, at lower concentrations (+)-10 and (+)-11
exhibited slightly weaker activity than the natural hormone (+)-
ABA. However, at the highest concentration tested (100 lM),
both analogues were slightly more potent inhibitors of corn cell
growth than (+)-ABA. The inhibitory activity of (+)-10 and (+)-
Fig. 6 Arabidopsis seed germination assay for (+)-8, (−)-8, (−)-1 and
(+)-1.
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for the (−)-enantiomer of ABA 1, which was only effective at
concentrations ≥3.33 lM.
The hydroxylated hormone analogues 10 and 11 show activity
in two physiological assays and will be valuable at the molecular
biological level for probing the roles of ABA catabolites in
processes regulated by ABA.
All three compounds also inhibited Arabidopsis thaliana seed
germination (Fig. 7). Interestingly, bicyclic ABA analogue (+)-
11 and (+)-ABA produced comparable effects although (+)-11
appeared to produce a slightly stronger inhibition of germination
than (+)-ABA at the lowest concentration tested (0.33 lM). The
weakest compound tested was (+)-10. This indicated that the
9ꢀ-derivative [ABA numbering] ((+)-11) possesses substantially
higher activity than the 8ꢀ-derivative ((+)-10). This unexpected
result is consistent to previous observations of very strong inhi-
bition of ABA 8ꢀ-hydroxylase enzyme activity by 9ꢀ-derivatives.21
Until recently, 9ꢀ-metabolites of (+)-ABA were not thought to
occur naturally, however (+)-9ꢀ-hydroxy ABA 6 has recently been
documented as a new ABA metabolite.11 Metabolite 6 has been
shown to be important in developing seeds of Brassica napus.25
The results described here suggest that natural and synthetic 9ꢀ-
ABA derivatives may have significant biological activity; even
more activity than 8ꢀ-ABA derivatives.
Experimental
General
FTIR spectra were recorded using KBr cells on a Perkin Elmer
1
Paragon 1000 instrument. H NMR and 13C were recorded on
a Bruker AM 500 MHz spectrometer. All NMR spectra were
obtained using CDCl3 as the solvent unless otherwise noted.
Chemical shifts (d) and coupling constants (J) are reported as if
they are first order. All peak assignments refer to the numbering in
structure 8. High-resolution mass spectra (HRMS) were recorded
in either electron impact (EI) mode, chemical ionization (CI) mode
or in negative ion electrospray mode using capillary voltage of
2.75 KV, counter electrode 35 V, collision energy (ELAB) of 14 V
and cell pressure of 1.0 × 10−3 mBar with argon. Mass spectra data
are reported in mass to charge units (m/z). Optical rotations were
obtained from a Perkin Elmer 141 polarimeter. Melting points
were measured on an Electrothermal 9300 melting point apparatus
and are not corrected.
Crystallographic data was collected at −100 ◦C on a Nonius
Kappa CCD diffractometer, using the COLLECT program.28 Cell
refinement and data reductions used the programs DENZO and
SCALEPACK.29 SIR9730 was used to solve the structure and
SHELXL9731 was used to refine the structure. XTAL3.732 was
used for molecular graphics. H atoms were placed in calculated
positions with Uiso constrained to be 1.2 times Ueq of the carrier
atom for methine, methylene and aromatic protons and 1.5 times
Ueq of the carrier atoms for methyl, N–H and O–H hydrogen
atoms.
Seed germination test
Fig. 7 Arabidopsis seed germination assay for (+)-10, (+)-ABA 1 and
(+)-11.
Arabidopsis thaliana (Columbia wild type) seed germination
inhibition studies were performed as described by Cutler et al.21
The treatments were performed in duplicate with 50 seeds per
plate and incubated at 24 ◦C with 16 h days and 8 h nights for the
duration of the test (7 d).
Conclusion
We have successfully synthesized a novel tetralone ABA analogue 8
through an efficient 7-step synthetic scheme using readily available
starting materials. This tetralone ABA analogue is significantly
more active than ABA in the two assays in which the compound
has been tested. The additional carbon atoms linking the C-3ꢀ and
C-7ꢀ of ABA in the tetralone analogue do not appear to affect
adversely the biological activity in either the seed germination
or the corn cell growth inhibition assays. It appears that the
binding sites in proteins that perceive or metabolize ABA can
accommodate the extra steric bulk of the tetralone analogue.
In a separate study on the structural requirements of an ABA
glycosyltransferase, (+)-8 has been found to be a better substrate
than (+)-ABA 1.26 The presence of the aromatic moiety also
has provided opportunities for the synthesis of tethered tetralone
ABA analogues, which are being used in identification of ABA
binding proteins.27 The tetralone analogue has potential as a plant
growth regulator for agriculture and horticulture applications.
Growth inhibition assay
Maize cell cultures were treated as described by Balsevich et al.9
The cultures were incubated on a rotary shaker for 4 d, and then
the cells were separated from the medium by vacuum filtration and
weighed immediately. The effect of ABA and the tetralone ABA
analogues on cell growth was determined at various concentrations
(0–10 lM) by calculating the percentage increase in fresh weight
[(final weight × 100/initial weight) − 100)]. Measurements were
performed in triplicate and average values were normalized to a
control (untreated) value of 100%.
Metabolism studies
Suspension cultures of maize (Zea mays L. CV. Black Mexican
Sweet) were maintained as described previously by Ludwig et al.20
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Isolation of hydroxylated tetralone ABA (+)-10. For each
treatment, twelve 250 mL flasks containing 100 mL of medium
were prepared. Eleven of these flasks contained medium to which
the relevant bicyclic ABA analogue had been introduced from
a 95 lM ethanolic solution to give a final concentration of 50–
100 lM. The remaining flask contained an equivalent amount
of ethanol to serve as an analogue-free control (C1). Ten of the
flasks containing analogue-infused media, along with C1, were
inoculated with cells that were 1 day post sub-culture. Typically,
2.0–3.0 g of cells was added to each flask. The remaining flask
served as a control to check for compound stability over the course
of the experiment (C2). Thus a control was in place to differentiate
between degradation products resulting from simple chemical
breakdown of the analogues (C2) and also to identify metabolites
of the cells which were not attributed to the metabolism of the ABA
analogues being studied (C1). The cell cultures were incubated in
the dark on a rotary shaker at 25 ◦C for 4 d. The media from
each control flask and from three of the test flasks (T1–T3) were
samples for analysis at 12 h intervals.
performed with semi-preparative ChiracelTM AS column (Daicel
Chemical Industries, Ltd) with the products eluting in iso-PrOH-
hexane (20:80). Spectroscopic characterization of metabolite from
the culture filtrate confirmed it as the bicyclic methyl ester (+)-40.
Chemical synthesis
2,2-Dimethyl-3,4-dihydro-2H-naphthalen-1-one (13)14. To
a
suspension of NaH (8.2 g, 0.34 mol) in THF (250 mL) in a one-liter
round bottomed flask, was added, 1-tetralone (10.0 g, 0.690 mol)
dissolved in dry THF (25 mL). After stirring the mixture for 10 min
at rt, methyl iodide (11.1 mL, 178 mmol) was added via a syringe.
The mixture was then heated on an oil bath to 40 ◦C for 30 min,
and stirring continued at rt until the starting material disappeared.
The reaction was monitored by TLC using ethyl acetate–hexane
(1 : 6) solvent mixture. The reaction was quenched by addition
of water (slowly and dropwise) to destroy excess sodium hydride.
The mixture was then extracted with ethyl acetate, washed with
water and dried over sodium sulfate. Evaporation of the solvent
yielded a brown oil. Column chromatography using silica gel with
EtOAc–hexane (1 : 6) afforded clean 2,2-dimethyl-1-tetralone 13
(10.8 g, 83%). IR (mmax): 2956, 1682, 1601 cm−1. 1H NMR: d 8.03
(d, 1H, J = 7.5 Hz, ArH-8), 7.45 (t, 1H, J = 7.5 Hz, ArH-7), 7.28
(t, 1H, J = 7.5 Hz, ArH-6), 7.21 (d, 1H, J = 7.5 Hz, ArH-5), 2.97
(t, 2H, J = 6.5 Hz, 2 H-4), 1.97 (t, 2H, J = 6.5 Hz, 2 H-3), 1.20 (s,
6H, 2 × CH3). HRMS (m/z) C12H14O requires: 174.1045; found:
174.1031.
At the indicated times, 115 mL samples were removed from
C1, C2, T1, T2 and T3. From each of these samples, 100 mL
was applied to a Sep PackTM C18 cartridge that had been pre-
conditioned with 1 mL methanol, followed by 1 mL H2O. After
the sample was adsorbed to the cartridge, it was washed with
5% methanol to remove salts and polar media components.
Finally, adsorbed metabolites were eluted with 1 mL methanol
and collected in microcentrifuge vials. The samples were then
◦
dried at 45 C in an Eppendorf VacufugeTM over a period of
(Z)-1-(5ꢀ -Hydroxy-3ꢀ -methylpent-3ꢀ -en-1ꢀ -ynyl)-2,2-dimethyl-
1,2,3,4-tetrahydronaphthalen-1-ol (15). (Z)-3-Methylpent-2-en-
4-yn-1-ol (5.0 g, 52 mmol) in dry THF (300 mL) was cooled to
−78 ◦C under an atmosphere of argon. n-Butyl lithium (70.0 mL,
1.6 M in hexanes, 112 mmol) was then added slowly, via syringe.
The mixture was allowed to stir at −78 ◦C for 45 min, after
which, 2,2-dimethyl-1-tetralone 13 (7.5 g, 43 mmol), dissolved
in dry THF (50 mL), was added. The mixture was stirred for a
further 15 min at −78 ◦C and then the ice bath was removed. The
reaction mixture was stirred at rt for a further 3 h, by which point,
the starting material had disappeared. The reaction was quenched
by addition of a saturated solution of ammonium chloride. The
mixture was stirred for 10 min and extracted with ethyl acetate
(3 × 150 mL), washed with water (2 × 200 mL) and dried over
anhydrous Na2SO4. Evaporation of the solvent yielded the desired
alcohol as a brown oil. Column chromatography of the brown oil
using silica gel with ethyl acetate–hexane (1 : 2) gave allylic alcohol
15 (6.1 g, 78%). IR (mmax): 3376 (br), 2921, 2358 (w), 2211 (w), 1694
(m), 1633 (m), 1602 (w) cm−1. 1H NMR: d 7.77 (m, ArH-8ꢀ), 7.20
(m, 2 ArH-6ꢀ and 7ꢀ), 7.08 (m, ArH-5ꢀ), 5.84 (t, J = 6.5 Hz, 1
H-2), 4.27 (d, J = 6.5 Hz, 2 H-1), 2.84 (m, 2 H-4ꢀ), 2.00 (ddd, J =
13.5, 2.0, 7.0 Hz, 1 H-3ꢀ), 1.88 (s, 3 H-6), 1.66 (ddd, J = 13.5, 1.0,
5.0 Hz, 1 H-3ꢀ), 1.15 (s, 3 H-9ꢀ/10ꢀ), 1.07 (s, 3 H-9ꢀ/10ꢀ). 13C NMR:
d 138.8, 135.7, 134.9, 129.0, 128.2, 127.9, 126.4, 120.7, 96.5, 84.4,
75.1, 61.4, 37.5, 31.2, 25.7, 23.9, 23.6, 23.2. HRMS (m/z) C18H22O2
requires: 269.1542 [M-1]−; found 269.1536.
3–4 h. The samples were re-dissolved in 50 mL methanol and
diluted to 100 mL with H2O. Samples were kept in the dark and
analyzed on a Supelco (Bellefonte, PA) SupelcosilTM LC-18 column
(3.3 cm × 4.6 mm i.d., 3 mm packing preceded by a Supelco C-
18, 2 cm × 4.6 mm guard column). The column was eluted at
1.5 mL min−1 with 1% aqueous HOAc–methanol (7 : 3) using an
◦
isocratic method at 45 C. The eluent was monitored at 262 nm
using a Hewlett Packard variable wavelength detector and diode
array detector referenced at 450 nm. The samples (10 lL each)
were injected with an autosampler. When possible, peaks in the
chromatograms were identified by comparing their retention times
with those of authentic standards. At the end of the culture period,
the cells were removed from all test flasks by filtration (Whatman
#1) and the filtrate frozen and stored (−20 ◦C) until processed for
metabolite and analogue isolation. After thawing, the filtrate was
adjusted to pH 2.3 prior to the extraction.
The filtrate was extracted with hexanes (3 × 250 mL) then
EtOAc (3 × 500 mL). The EtOAc fractions were pooled and
extracted with 5% NaHCO3 (2 × 330 mL). The pH of the aqueous
layer was adjusted to 2.4 and then extracted with EtOAc (3 ×
330 mL). The combined organic fractions were washed with
saturated NaCl (150 mL), dried over anhydrous Na2SO4, filtered
and the solvent removed in vacuo to give a crude extract. The
extract was dissolved in 1 mL methanol then further purified using
preparative HPLC (PartisilTM 10 ODS-2 M9/25 column preceded
by a SuperguardTM C18 2 cm × 4.6 i.d. guard column) with 1%
aqueous HOAc–acetonitrile (4 : 6). Combined fractions of each
peak from successive runs were pooled and the solvent removed
in vacuo.
Methyl (2Z,4E)-(1ꢀRS)-5-(2ꢀ,2ꢀ-dimethyl-1ꢀ-hydroxy-1ꢀ,2ꢀ,3ꢀ,4ꢀ-
tetrahydronaphthalen-1ꢀ -yl)-3-methylpenta-2,4-dienoate
(17).
The allylic alcohol 15 (6.0 g, 22.1 mmol) in dry THF was cooled
◦
R
The methyl ester derivatives of each of the samples were
made by treatment with CH2N2–etherate. Further purification was
to −78 C and RedAlꢀ (13.7 ml, 44.2 mmol) added dropwise via
◦
syringe. The reaction mixture was stirred at −78 C for 1 h and
1406 | Org. Biomol. Chem., 2006, 4, 1400–1412
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The Royal Society of Chemistry 2006
©

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the allowed to warm up to 0 ◦C and stirred for a further 2 h.
The reaction was quenched by slow addition of water (100 mL)
and extracted with diethyl ether (2 × 200 mL). The organic phase
was washed with water (2 × 200 mL) and dried over anhydrous
Na2SO4. Evaporation of solvent left a crude brown oil of the
allylic alcohol 16 (6.05 g), which was carried through to the next
stage without any further purification. The crude allylic alcohol
16 (6.05 g, 22.2 mmol) was dissolved in dry acetone (250 mL) and
manganese dioxide (38.7 g, 445 mmol) was added. The mixture
was stirred at rt for 3 h, after which all the starting material had
disappeared. The black suspension was then filtered through a
23.4, 21.7. HRMS (m/z) C19H22O4 requires: 314.1518; found:
314.1521. The enantiomers of ester 18 were resolved by chiral
HPLC (ChiralcelTM AS column (10 × 250 mm; Daicel Chemical
Industries, Ltd., iso-PrOH–hexane, 3 : 97) and had the following
optical rotations: [a]2D5 +247 (c 1.2, CHCl3) (retention time
12.5 min) and −243 (c 1.0, CHCl3) (retention time 15.8 min) for
(+)-18 and (−)-18, respectively.
(2Z,4E)-(1ꢀS)-5-(2ꢀ,2ꢀ -Dimethyl-1ꢀ -hydroxy-4ꢀ -oxo-1ꢀ,2ꢀ,3ꢀ,4ꢀ-
tetrahydronaphthalen-1ꢀ-yl)-3-methylpenta-2,4-dienoic acid (+)-8).
A mixture of ester (+)-18 (0.05 g, 0.159 mmol) in MeOH (4 mL)
and 1.0 M KOH (4 mL) was stirred at 45 ◦C for 2 h, by which
point, all the starting material had disappeared. The solvent was
evaporated at reduced pressure, the aqueous layer acidified to pH 3
with 10% Cl and extracted with ethyl acetate (3 × 50 mL). The
combined organic extracts were dried over anhydrous Na2SO4,
and concentrated to provide acid (+)-8 (0.037 g, 76%). [a]2D5 +193
R
bed on Celiteꢀ. Evaporation of solvent left a clear brown oil of
the aldehyde (4.29 g), which was carried through to the next stage
1
without any further purification. H NMR of the crude mixture
showed the presence of an aldehyde proton. To the aldehyde
(4.29 g, 15.9 mmol), dissolved in methanol (150 mL), were added,
manganese dioxide (27.7 g, 318.0 mmol), sodium cyanide (2.80 g,
57.2 mmol) and glacial acetic acid (1.05 g, 17.5 mmol). The
mixture was stirred at rt for 4 h, after which all the starting
material had disappeared. The suspension was filtered over a
1
(c 1.0, MeOH). IR (mmax): 3606–2488, 3453,1685, 1598 cm−1. H
NMR: d 8.02 (dd, J = 7.8, 1.2 Hz, ArH-5ꢀ), 7.74 (d, J = 16.0 Hz,
1 H-4), 7.52–7.59 (m, 2 ArH-7ꢀ and 8ꢀ), 7.38–7.41 (m, ArH-6ꢀ/7ꢀ),
6.42 (d, J = 16.0 Hz, 1 H-5), 5.72 (s, 1 H-2), 2.80 (d, J = 17.0 Hz,
1 H-3ꢀ), 2.56 (d, J = 17.0 Hz, 1 H-3ꢀ), 2.02 (s, 3 H-6), 1.08 (s, 3
H-9ꢀ/10ꢀ), 1.06 (s, 3 H-9ꢀ/10ꢀ). 13C NMR: d 197.4, 171.0, 151.8,
145.6, 139.2, 134.5, 130.9, 128.4, 128.2, 127.2, 126.7, 117.7, 78.4,
60.4, 49.7, 41.1, 24.3, 23.4, 21.4. HRMS (m/z) C18H20O4 requires:
300.1362; found: 300.1351.
R
bed of Celiteꢀ and washed with methanol (3 × 100 mL). The
combined filtrate was then concentrated under vacuum to yield a
light brown solid. Water (150 mL) was then added to the crude
solid and then extracted with ethyl acetate (3 × 200 mL). The
organic phase was washed with water (3 × 100 mL) and dried over
anhydrous Na2SO4. Evaporation of solvent yielded a brown oil.
Column chromatography using silica gel and 25% ethyl acetate
in hexane gave methyl ester 17 (3.3 g, 49%) over the three steps.
IR (mmax): 3500, 2924, 1715, 1633, 1601 cm−1. 1H NMR: d 7.79 (d,
J = 16.0 Hz, 1 H-4), 7.36 (dd, J = 6.5 and 1.0 Hz, ArH-8ꢀ), 7.16
(m, 2 ArH), 7.11 (m, 1 ArH), 6.30 (d, J = 16.0 Hz, 1 H-5), 5.68
(s, 1 H-2), 3.67 (s, 3 H, CO2CH3), 2.86 (t, J = 7.0 Hz, 2 H-4ꢀ),
1.98 (s, 3 H-6), 1.89 (dt, J = 14.0, 7.0 Hz, 1 H-3ꢀ), 1.68 (dt, J =
Methyl (2Z,4E,4ꢀE)-(1ꢀS,2ꢀꢀR)-5-[2ꢀ,2ꢀ-dimethyl-1ꢀ-hydroxy-4ꢀ-
(2ꢀꢀ -methoxymethylpyrrolidin-1ꢀꢀ -ylimino)-1ꢀ,2ꢀ,3ꢀ,4ꢀ -tetrahydrona-
phthalen-1ꢀ-yl]-3-methylpenta-2,4-dienoate (20). To a mixture of
ester 18 (42 mg, 0.13 mmol), RAMP (20 lL, 0.14 mmol), PTSA
(9.2 mg, 0.048 mmol) in dry toluene (2 mL) was heated at 110–
112 ◦C for 1 d. The reaction mixture was diluted with CH2Cl2
after it was cooled to rt. The organic layer was washed with sat.
NaHCO3, dried, and evaporated to give a residue. The residue
was separated by flash column chromatography (10–40 l silica gel
was used with 20% ethyl acetate–hexane) to provide hydrazone
20 (22.2 mg, 40%), [a]2D5 −425 (c 1.3, CH2Cl2) and an inseparable
mixture of starting material and the other diastereomeric product
(25.3 mg). IR (mmax): 3457, 2965, 1714, 1634, 1601 cm−1. 1H NMR:
d 8.11 (m, 1H, Ar–H), 7.96 (d, 1H, J = 16.0 Hz, H-4), 7.23 (m,
3H, Ar–H), 6.31 (d, 1H, J = 16.0 Hz, H-5), 5.69 (s, 1H, H-2), 3.50
(m, 2H, CH2OCH3), 3.35 (m, 1H, H-5ꢀꢀ), 3.32 (s, 3H, OCH3), 3.29
(m, 1H, H-2ꢀꢀ), 2.72 (d, 1H, J = 16.0 Hz, H-3ꢀ), 2.65 (d, 1H, J =
16.0 Hz, H-3ꢀ), 2.51 (q, 1H, J = 8.5 Hz, H-5ꢀꢀ), 2.03 (m, 1H, H-3ꢀꢀ),
2.00 (s, 3H, 6-CH3), 1.85 (m, 2H, H-4ꢀꢀ), 1.71 (m, 1H, H-3ꢀꢀ), 0.99
(s, 3H, 10ꢀor 9ꢀ-CH3), 0.88 (s, 3H, 9ꢀor 10ꢀ-CH3). 13C NMR: d 166.6,
154.9, 150.0, 140.3, 139.7, 132.4, 129.4, 127.9, 127.8, 127.2, 124.8,
117.2, 77.7, 75.6, 67.0, 59.2, 54.6, 51.1, 39.1, 38.6, 26.8, 24.2, 24.0,
22.9, 21.3. HRMS (m/z) C25H34N2O4 requires: 427.2596 [M + 1]+;
found 427.2597.
14.0, 7.0 Hz, 1 H-3ꢀ), 1.00 (s, 3 H-9ꢀ/10ꢀ), 0.96 (s, 3 H-9ꢀ/10ꢀ). 13
C
NMR: d 166.7, 150.3, 141.4, 140.4, 135.7, 128.9, 128.2, 127.3,
126.6, 126.4, 117.0, 78.1, 51.0, 37.2, 33.0, 25.9, 24.1, 23.1, 21.3.
HRMS (m/z) C19H24O3 requires: 300.1725; found: 300.1721.
Methyl (2Z,4E)-(1ꢀS)-5-(2ꢀ,2ꢀ -dimethyl-1ꢀ - hydroxy - 4ꢀ -oxo-
1ꢀ,2ꢀ,3ꢀ,4ꢀ-tetrahydronaphthalen-1ꢀ-yl)-3-methylpenta-2,4-dienoate
((+)-18). To the ester 17 (3.0 g, 10 mmol) dissolved in benzene
(100 mL), were added, pyridinium dichromate (5.64 g, 30 mmol)
and tert-butyl hydroperoxide (1.35 g, 15 mmol). The mixture
was stirred at rt for 4 h. Diethyl ether (50 mL) was added to the
reaction mixture and stirring continued for a further 30 min. The
R
mixture was then filtered through a bed of Celiteꢀ and washed
with diethyl ether (3 × 25 mL). The combined organic filtrate
was then concentrated in vacuo, leaving a brown oil. Column
chromatography using silica gel with 25% ethyl acetate in hexane
afforded the unreacted starting material (1.20 g) with Rf 0.5 and
the desired ester 18 (1.48 g, 78%, based on amount of starting
material consumed). IR (mmax): 3457, 3067, 2962, 1722, 1682, and
Hydrolysis of hydrazone 20
1
1599 cm−1. H NMR: d 8.03 (dd, J = 1.0, 8.0 Hz, ArH-5ꢀ), 7.82
(d, J = 16.0 Hz, 1 H-4), 7.56 (m, 2 ArH-6ꢀ and 8ꢀ), 7.42 (t, J =
7.0 Hz, ArH-7ꢀ), 6.35 (d, J = 16.0 Hz, 1 H-5), 5.72 (s, 1 H-2),
3.66 (s, 3 H, CO2CH3), 2.80 (d, J = 17.0 Hz, 1 H-3ꢀ), 2.56 (d, J =
17.0 Hz, 1 H-3ꢀ), 1.98 (s, 3 H-6), 1.07 (s, 3 H-9ꢀ/10ꢀ), 1.06 (s, 3
H-9ꢀ/10ꢀ). 13C NMR: d 197.3, 166.4, 149.4, 145.7, 138.5, 134.4,
130.9, 128.1 (2), 127.2, 126.7, 118.1, 78.2, 51.1, 49.7, 41.0, 24.3,
A mixture of RAMP–hydrazone 20 (12.0 mg, 0.028 mmol) in
hexane (2 mL) and CH2Cl2 (0.1 mL) saturated oxalic acid (0.5 mL)
was stirred at rt for 3 d. The reaction mixture was extracted with
CH2Cl2, washed with saturated NaHCO3, dried and evaporated
to give a residue. The residue was purified by flash column
chromatography (25% ethyl acetate–hexane) to afford a product
This journal is
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Org. Biomol. Chem., 2006, 4, 1400–1412 | 1407
©

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1
which from H NMR and the retention time from chiral HPLC
NaHCO3. The ethanol was then removed in vacuo to give a residue,
which was diluted with CH2Cl2 and washed with water, dried and
concentrated to provide a crude product. The crude product was
purified by column chromatography on silica gel (10–40 l) using
30% ethyl acetate in hexane to give the pure dansyl hydrazone
23 (62.4 mg, 79%) as a yellow powder. The yellow powder was
then recrystallized from hexane–ethyl acetate (4 : 1) to give the
(Chiralpak AS column, 250 × 10 mm, Diacel Chemical Industries
Ltd, Japan) was confirmed as the ester (+)-17 (5.3 mg, 61%), [a]2D5 +
255 (c 0.53, CHCl3).
(4E,1ꢀE,3ꢀZ)-(1S,2ꢀꢀR)-1-(5ꢀ -Hydroxy-3ꢀ -methylpenta-1ꢀ,3ꢀ-
dienyl)-4-(2ꢀꢀ -methoxymethylpyrrolidin-1ꢀꢀ -ylimino)-2,2-dimethyl-
1,2,3,4-tetrahydronaphthalen-1-ol (21). To
a
suspension of
◦
crystalline product: Mp: 110.1–114.9 C (decomposition), [a]D25
LiAlH4 (63.7 mg, 1.68 mmol) in anhydrous ether (15 mL) was
added hydrazone 20 (113 mg, 0.28 mmol) at rt and the mixture
stirred at rt for 3 h. The reaction was quenched with a drop of
water and then more water added. The mixture was acidified
with 3 N HCl to pH 4.0 and EtOAc added. The mixture was
then stirred for 20 min and then extracted with EtOAc, dried,
and concentrated to give a crude product which was purified by
column chromatography on silica gel, using 30% ethyl acetate–
hexane followed by 50% ethyl acetate–hexane) to provide the
pure hydrazone alcohol 21 (86.9 mg, 78%). IR (mmax) 3418, 2965,
2871 cm−1. 1H NMR: d 8.11 (m, 1H, Ar–H), 7.25 (m, 3H, Ar–H),
6.83 (d, 1H, J = 15.5 Hz, H-4), 5.96 (d, 1H, J = 15.5 Hz, H-5),
5.56 (t, 1H, J = 7.0 Hz, H-2), 4.17 (m, 2H, H-1), 3.52 (m, 2H,
CH2OCH3), 3.35 (m, 1H, H-5ꢀꢀ), 3.31 (s, 3H, OCH3), 3.29 (m, 1H,
H-2ꢀꢀ), 2.67 (s, 2H, H-3ꢀ), 2.51 (m, 1H, H-5ꢀꢀ), 2.03 (m, 1H, H-
3ꢀꢀ), 1.86 (m, 2H, H-4ꢀꢀ), 1.86 (s, 3H, 6-CH3), 1.72 (m, 1H, H-3ꢀꢀ),
0.97 (s, 3H, 10ꢀor 9ꢀ-CH3), 0.88 (s, 3H, 9ꢀor 10ꢀ-CH3). 13C NMR: d
155.1, 140.7, 134.4, 133.5, 132.5, 129.3, 128.4, 127.8, 127.1, 126.8,
124.8, 77.8, 75.6, 66.9, 59.2, 58.4, 54.6, 39.2, 38.4, 26.8, 24.2, 24.0,
22.9, 20.8. HRMS (m/z) C24H35N2O3 requires: 399.2647 [M + 1]+;
found: 399.2656.
−306 (c 0.38, CH2Cl2). IR (mmax): 3513, 3214, 3059, 2960, 2871,
1689 (w), 1610, 1574 cm−1. 1H NMR: d 8.54 (d, 1H, J = 7.5 Hz, Ar–
H), 8.35 (m, 2H, Ar–H), 8.24 (br s,1H, NH), 8.12 (m, 1H, Ar–H),
7.91 (d, 1H, J = 10.0 Hz, H-1), 7.53 (m, 2H, Ar–H), 7.25 (m, 3H,
Ar–H), 7.15 (d, 1H, J = 7.5 Hz, Ar–H), 6.86 (d, 1H, J = 15.0 Hz,
H-4), 6.04 (d, 1H, J = 15.0 Hz, H-5), 5.93 (d, 1H, J = 10.0 Hz,
H-2), 3.52 (m, 2H, CH2OCH3), 3.38 (m, 1H, H-5ꢀꢀ), 3.32 (m, 1H,
H-2ꢀꢀ), 3.32 (s, 3H, OCH3), 2.86 (s, 6H, N(CH3)2), 2.66 (m, 2H,
H-3ꢀ), 2.54 (m, 1H, H-5ꢀꢀ), 2.02 (m, 1H, H-3ꢀꢀ), 1.88 (m, 2H, H-4ꢀꢀ),
1.88 (s, 3H, 6-CH3), 1.70 (m, 1H, H-3ꢀꢀ), 0.95 (s, 3H, 10ꢀor 9ꢀ-CH3),
0.88 (s, 3H, 9ꢀor 10ꢀ-CH3). 13C NMR: d 154.4, 151.9, 145.0, 141.0,
140.3, 136.1, 133.6, 132.5, 131.14, 131.08, 129.82, 129.75, 129.4,
128.4, 128.1, 127.0, 126.2, 124.8, 124.4, 123.3, 118.9, 115.2, 78.0,
75.6, 66.7, 59.0, 54.8, 45.4, 39.2, 38.6, 26.7, 24.2, 24.0, 22.9, 21.0.
HRMS (m/z) C36H46N5O4S requires: 644.3270 [M + 1]+; found:
644.3265. Single crystals of C36H45N5O4S 23 were recrystalized
from methanol, mounted in inert oil and transferred to the cold
gas stream of the diffractometer.
Crystal structure determination of compound 23§
Crystal data. C38H53N5O6S, M = 707.91, orthorhombic, a =
(2Z,4E,4ꢀE)-(1ꢀS,2ꢀꢀR)-5-[1ꢀ-Hydroxy-4ꢀ-(2ꢀꢀ-methoxymethylpyrro-
lidin-1ꢀꢀ -ylimino)-2ꢀ,2ꢀ -dimethyl-1ꢀ,2ꢀ,3ꢀ,4ꢀ -tetrahydronaphthalen-
1ꢀ-yl]-3-methylpenta-2,4-dienal (22). A mixture of hydrazone
alcohol 21 (69 mg, 0.17 mmol) and MnO2 (300.8 mg, 3.46 mmol)
in acetone (5 mL) was stirred at RT for 16 h. The reaction mixture
˚
11.2340(2), b = 16.3940(2), c = 20.9930(3) A, U = 3866.28(10)
3
−1
˚
A , T = 173(2) K, space group P212121, Z = 4, l = 0.134 mm ,
32824 reflections measured, 4913 unique (Rint = 0.0637) which
were used in all calculations. The final wR2 was 0.0992 (all data).
The absolute configuration was known from the derivatization
and was not independently determined. There are two solvent
methanol molecules per asymmetric unit in the crystal. Both are
almost as well ordered as the major molecule and both are involve
in strong H-bonding schemes with one another and the major
molecule. Two atoms are showing asymmetric thermal ellipsoids
(O4 and C31), one being an ether oxygen and the other a terminal
methyl group. The disorder is not sufficiently severe so as to require
modelling of the disorder. Both groups would be expected to have
high vibrational motion. Most of the checkCIF errors are related
to the fact that the absolute configuration was not determined
using Friedel mates, because the configuration of the derivative
was known.
R
was filtered over a bed of Celiteꢀ and washed with acetone. The
combined filtrates and washings were evaporated to give a residue,
which was purified by column chromatography on silica using
30% ethyl acetate–hexane to provide the hydrazone aldehyde 22
(54.4 mg, 79%) and [a]D25 −533 (c 0.43, CH2Cl2). IR (mmax): 3429,
3060, 2965, 2873, 1666, 1632 cm−1. 1H NMR: d 10.2 (d, 1H, J =
8.0 Hz, H-1), 8.16 (m, 1H, Ar–H), 7.58 (d, 1H, J = 15.5 Hz, H-4),
7.27 (m, 3H, Ar–H), 6.38 (d, 1H, J = 15.5 Hz, H-5), 5.90 (d, 1H,
J = 8.0 Hz, H-2), 3.54 (m, 2H, CH2OCH3), 3.40 (m, 1H, H-5ꢀꢀ),
3.34 (s, 3H, OCH3), 3.32 (m, 1H, H-2ꢀꢀ), 2.71 (m, 2H, H-3ꢀ), 2.55
(m, 1H, H-5ꢀꢀ), 2.10 (s, 3H, 6-CH3), 2.07 (m, 1H, H-3ꢀꢀ), 1.89 (m,
2H, H-4ꢀꢀ), 1.75 (m, 1H, H-3ꢀꢀ), 1.03 (s, 3H, 10ꢀor 9ꢀ-CH3), 0.94 (s,
3H, 9ꢀor 10ꢀ-CH3). 13C NMR: d 190.4, 154.0, 153.6, 140.8, 139.7,
132.6, 129.5, 128.9, 128.3, 127.0, 125.7, 125.1, 78.0, 75.6, 67.0,
59.2, 54.8, 39.2, 38.7, 26.8, 24.3, 24.0, 23.0, 21.6. HRMS (m/z)
C24H33N2O3 requires: 397.2458 [M + 1]+; found: 397.2490.
(1E,2ꢀZ,4ꢀE,4ꢀꢀE)-(1ꢀꢀS,2ꢀꢀꢀR)-5-(Dimethylamino)-Nꢀ -{5ꢀ -[1ꢀꢀ-
hydroxy - 4ꢀꢀ -(2ꢀꢀꢀ -(methoxymethyl)pyrrolidin-1ꢀꢀꢀ -ylimino)-2ꢀꢀ,2ꢀꢀ -
dimethyl-1ꢀꢀ,2ꢀꢀ,3ꢀꢀ,4ꢀꢀ -tetrahydronaphthalen-1ꢀꢀ -yl]-3ꢀ -methylpenta-
2ꢀ,4ꢀ-dienylidene}-naphthalene-1-sulfonohydrazide (23). A mix-
ture of the hydrazone aldehyde 22 (48.9 mg, 0.12 mmol), dansyl
hydrazine (32.8 mg, 0.12 mmol) and trichloroacetic acid (8.6 mg,
0.053 mmol) in ethanol (2 mL) was heated at 75 ◦C for 5 min.
The reaction was quenched by addition of several drops of sat.
2-Methyl-4,4-ethylenedioxynaphthalen-1-one (25). 2-Methyl-
1-naphthol 24 (5.0 g, 31.6 mmol) dissolved in ethylene glycol
(100 mL) was added to a round bottomed flask (500 mL),
containing iodobenzene diacetate (21.4 g, 66.4 mmol) dissolved
in ethylene glycol (100 mL) and stirred at rt for 4 h. Reaction
was quenched by addition of H2O (50 mL) followed by extraction
with diethyl ether (3 × 150 mL). The organic phase was washed
with saturated NaCl solution (2 × 200 mL), dried over anhydrous
Na2SO4 and dried in vacuo. Flash chromatography using silica gel
§ CCDC reference number 278290. For crystallographic data in CIF
format see DOI: 10.1039/b509193d
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with 50% ether in hexane yielded 25 (4.4 g, 64%). Mp: 125.1–
125.9 ◦C IR (mmax): 3290, 3074, 2984, 2910, 1658 cm−1. 1H NMR:
d 8.05 (dt, 1H, J = 8.0, 1.0 Hz, Ar–H), 7.57 (m, 2H, Ar–H), 7.46
(ddd, 1H, J = 3.0, 5.5, 8.0 Hz, Ar–H), 6.62 (q, 1H, J = 1.5 Hz,
H-3), 4.33 (m, 2H), 4.25 (m, 2H), 1.99 (d, 3H, J = 1.5 Hz)).
13C NMR: d 184.6, 141.1, 138.4, 135.7, 132.9, 130.9, 129.4, 126.5,
126.3, 100.2, 65.8, 16.0. HRMS (m/z) C13H12O3 requires: 216.0786;
found: 216.0790.
(2Z,4E)-(1ꢀS,2ꢀR)-5-(2ꢀ -Ethynyl-1ꢀ -hydroxy-2ꢀ -methyl-4ꢀ -oxo-
1ꢀ,2ꢀ,3ꢀ,4ꢀ -tetrahydronaphthalen-1ꢀ -yl)-3-methylpenta-2,4-dienoic
acid ((+)-31). A mixture of ester (+)-30 (0.013 g, 0.0401 mmol)
in MeOH (1 mL) and 2.0 M KOH (1 mL) was stirred at 25 ◦C for
2 h, by which point, all the starting material had disappeared.
The solvent was evaporated at reduced pressure, the aqueous
layer acidified to pH 3 with 10% HCl and extracted with ethyl
acetate (3 × 50 mL). The combined organic extracts were dried
over anhydrous Na2SO4, and concentrated to provide acid (+)-31
Methyl
(2Z,4E)-(1ꢀRS)-5-(1ꢀ-hydroxy-2ꢀ-methyl-4ꢀ-oxo-1ꢀ,4ꢀ-
(0.0101 g, 81%) as a white powder (was not re-crystallized). [a]D25
+
dihydronaphthalen-1ꢀ-yl)-3-methylpenta-2,4-dienoate (29). Com-
pound 25 (2.0 g, 9.3 mmol) converted to ketal ester 28 in the same
manner as compound 13 was converted methyl ester 17. To the
above brown oil (ketal ester 28, 1.4 g) in THF (50 mL) in an ice
bath, was added 10% HCl (2 mL) and mixture stirred for 1 h, after
which all starting material had disappeared. Water (20 mL) was
added to mixture and extracted with diethyl ether (3 × 100 mL).
The organic phase was washed with saturated NaCl (100 mL)
and dried over anhydrous Na2SO4 and dried in vacuo. Flash
chromatography using silica gel with 3 : 1 (diethyl ether–hexane)
mixture afforded enone 29 (1.2 g, 75% over five steps) as white
crystals. Mp = 147–148 ◦C (EtOAc). IR (mmax): 3402, 3070, 2951,
200 (c 1.0, MeOH). IR (mmax): 3600–2400, 3500, 3284, 2979, 1682,
1598 cm−1. 1H NMR (CD3OD): d 7.96 (dd, 1H, J = 1.5, 8.0 Hz,
Ar–H), 7.70 (d, 1H, J = 7.5 Hz, Ar–H), 7.65 (dt, 1H, J = 1.5,
7.5 Hz, Ar–H), 7.52 (d, 1H, J = 16.0 Hz, H-4), 7.43 (dt, 1H, J =
1.5, 8.0 Hz, Ar–H), 6.37 (d, 1H, J = 16.0 Hz, H-5), 5.69 (s, 1H,
H-2), 2.90 (d, 1H, J = 17.0 Hz, H-3ꢀ), 2.81 (d, 1H, J = 17.0 Hz, H-
3ꢀ), 2.39 (s, 1H, alkyne), 1.98 (s, 3H, H-6), 1.43 (s, 3H, H-10ꢀ). 13
C
NMR (CD3OD): d 197.4, 169.3, 150.6, 148.7, 137.8, 135.5, 132.5,
131.0, 128.9, 128.7, 127.2, 120.1, 88.0, 78.7, 73.5, 44.7, 34.3, 23.4,
21.1. HRMS (m/z) C19H17O4 requires: 309.1127 [M-1]−; found:
309.1117.
2-Hydroxymethylene-3,4-dihydro-2H-naphthalen-1-one (32)22.
Hydroxymethylene-1-tetralone 32 (18.3 g, 92%) was prepared from
a-tetralone 12 (16.6 g, 114 mmol). IR (mmax): 3644–3200, 3025, 2846,
1
1710, 1657, 1599 cm−1. H NMR: d 8.09 (d, 1H, J = 15.9 Hz),
8.01 (d, 1H, J = 7.8 Hz), 7.64 (d, 1H, J = 7.8 Hz), 7.54 (t, 1H,
J = 7.8 Hz), 7.39 (t, 1H, J = 7.8 Hz), 6.21 (s, 1H), 5.75 (d, 1H,
J = 15.9 Hz), 5.69 (s, 1H), 3.67 (s, 3H), 2.08 (s, 3H), 1.87 (s, 3H).
13C NMR: d 183.9, 166.4, 160.6, 149.6, 145.8, 138.4, 133.1, 129.4,
128.2, 127.5, 126.8, 126.4, 126.1, 118.2, 73.4, 51.2, 20.9, 18.6.
HRMS (m/z) C18H18O4 requires: 298.1205; found: 298.1190.
1
1602 cm−1. H NMR: d 14.61 (s, 1H), 8.20 (s, 1H), 7.93 (d, 1H,
J = 7.7 Hz, Ar–H), 7.41 (t, 1H, J = 7.5 Hz, Ar–H), 7.31 (t, 1H,
J = 7.5 Hz, Ar–H), 7.20 (d, 1H, J = 7.5 Hz, Ar–H), 2.86 (t, 3H,
J = 7.4 Hz, CH3), 2.54 (t, 3H, J = 7.4 Hz, CH3). HRMS (m/z)
C11H10O2 requires: 174.0681; found: 174.0685.
Methyl (2Z,4E)-(1ꢀS,2ꢀR/1ꢀR,2ꢀS)-5-(2ꢀ-ethynyl-1ꢀ-hydroxy-2ꢀ-
methyl-4ꢀ-oxo-1ꢀ,2ꢀ,3ꢀ,4ꢀ-tetrahydronaphthalen-1ꢀ-yl)-3-methylpenta-
2,4-dienoate (30). Enone 29 (1.00 g, 3.35 mmol) in dry THF
(15 mL) was added dropwise to a cold (0 ◦C) solution of
ethynylmagnesium bromide (33.6 mL, 0.5 M in diethyl ether) in
THF (15 mL). After stirring for 6 h, the reaction mixture was
quenched by the addition of saturated NH4Cl (25 mL), followed
by extraction with diethyl ether (3 × 100 mL). The organic phase
was dried over anhydrous MgSO4, filtered through Celite and
dried in vacuo, leaving a yellow oil (1.18 g). Flash chromatography
using silica gel with 20% ethyl acetate in hexane yielded methyl
8ꢀ-acetylene tetralone ABA 30 (0.633 g, 58%) as a light yellow
solid. IR (mmax): 3481, 3291, 2080, 1981, 1691, 1636, 1601 cm−1.
1H NMR: d 8.03 (dd, 1H, J = 1.0, 8.0 Hz, H-5ꢀ), 7.70 (d, 1H, J =
8.0 Hz, H-8ꢀ), 7.62 (d, 1H, J = 16.0 Hz, H-4), 7.61 (dt, 1H, J =
1.0, 8.0 Hz, H-7ꢀ), 7.42 (dt, 1H, J = 1.0, 8.0 Hz, H-6ꢀ), 6.15 (d,
1H, J = 16.0 Hz, H-5), 5.68 (s, 1H, H-2), 3.61 (s, 3H, OCH3),
2.93 (d, 1H, J = 17.1 Hz, H-3ꢀ), 2.82 (s, 1H, OH), 2.77 (d, 1H,
J = 17.1 Hz, H-3ꢀ), 2.11 (s, 1H, alkyne), 1.93 (s, 3H, H-6), 1.42 (s,
3H, H-10ꢀ). 13C NMR: d 194.6, 166.1, 148.8, 146.5, 134.7, 134.5,
130.9, 130.1, 128.2, 127.4, 126.6, 118.9, 86.1, 77.2, 73.7, 51.1,
48.2, 43.9, 22.9, 20.9. HRMS (FAB+, m/z) C20H21O4 requires
325.1440 [M + 1]+; found: 325.1401. The enantiomers of ester
30 were resolved by chiral HPLC (ChiralcelTM OD column (10 ×
250 mm; Daicel Chemical Industries, Ltd., iso-PrOH–hexane,
20 : 80, 2.0 mL min−1) and had the following optical rotations:
[a]2D5 + 265 (c 0.88, CHCl3) (retention time 18 min) and −288
(c 1.8, CHCl3) (retention time 26 min) for (+)-30 and (−)-30,
respectively.
2-Methyl-1-oxo-1,2,3,4-tetrahydro-naphthalene-2-carbaldehyde
(33)23. A mixture of 2-hydroxymethylene-1-tetralone 32 (18.0 g,
102 mmol), CH3I (7.0 mL, 113 mmol) and KOH (6.3 g, 113 mmol)
in anhydrous 1,2-dimethoxyethane (DME) (200 mL) was stirred
at rt under argon atmosphere for 12 h. The precipitate (KI) was
filtered off and the filtrate concentrated in vacuo. The residue was
redissolved in diethyl ether (500 mL), washed with H2O (2 ×
150 mL) and dried over anhydrous Na2SO4. Concentration of
the ethereal solution and column chromatography of the residue
using silica gel and 15% ethyl acetate in hexane afforded the
aldehyde 33 (8.2 g, 44%) and 2-methoxymethylene-3,4-dihydro-
2H-naphthalen-1-one as a by-product (8.9 g, 46%). IR (mmax): 1732,
1
1681, and 1602 cm−1. H NMR: d 9.64 (s, 1H, CHO), 7.94 (d,
1H, J = 7.8 Hz, Ar–H), 7.41 (t, 1H, J = 7.4 Hz, Ar–H), 7.22
(t, 1H, J = 7.4 Hz, Ar–H), 7.15 (d, 1H, J = 7.8 Hz, Ar–H),
2.93 (m, 2H, CH2), 2.40 (m, 1H, CH2), 1.92 (m, 1H, CH2), 1.31
(s, 3H, CH3),. HRMS (m/z) C12H12O2 requires: 188.0837; found:
188.0825.
2-Methoxymethylene-3,4-dihydro-2H -naphthalen-1-one. 1H
NMR: d 8.01 (d, 1H, J = 7.8 Hz, Ar–H), 7.50 (s, 1H, CH3), 7.39
(t, 1H, J = 7.3 Hz, Ar–H), 7.28 (t, 1H, J = 7.3 Hz, Ar–H), 7.19 (d,
1H, J = 7.5 Hz, Ar–H), 3.87 (s, 3H, CH3), 2.85 (t, 2H, J = 6.5 Hz,
CH2), 2.68 (t, 2H, J = 6.5 Hz, CH2). HRMS (m/z) C12H12O2
requires: 188.0837; found 188.0839.
2-Dimethoxymethyl-2-methyl-3,4-dihydro-2H-naphthalen-1-one
(34). To a solution of aldehyde 33 (4.0 g, 21.3 mmol) in reagent
grade MeOH (200 mL), was added, 0.25 g NH4Cl and reaction
mixture stirred at 65 ◦C for 3 h. The solvent was evaporated
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in vacuo and the resulting residue taken up in ethyl acetate
(350 mL), washed with H2O (2 × 100 mL) and dried over
anhydrous Na2SO4. Column chromatography using silica gel with
14% ethyl acetate in hexane afforded the keto acetal 34 (3.64 g,
73%) as a yellow oil. IR (mmax): 3340, 2934, 2830, 1679, 1601 cm−1.
1H NMR: d 7.98 (d, 1H, J = 7.8 Hz, Ar–H), 7.39 (t, 1H, J =
7.3 Hz, Ar–H), 7.23 (t, 1H, J = 7.3 Hz, Ar–H), 7.17 (d, 1H, J =
7.8 Hz, Ar–H), 4.67 (s, 1H, CH), 3.51 (s, 3H, OCH3), 3.43 (s,
3H, OCH3), 2.91 (m, 2H, CH2), 2.34 (m, 1H, CH2), 1.88 (m, 1H,
CH2), 1.14 (s, 3H, CH3). 13C NMR: d 200.1, 143.6, 133.0, 131.7,
127.5, 127.1, 126.3, 110.4, 58.6, 58.5, 50.4, 26.6, 24.9 and 19.0.
HRMS (m/z) C14H18O3 requires: 234.1256; found: 234.1252.
(0.566 g, 1.57 mmol) yielded the desired compound 38 (0.260 g,
44%) and recovered starting material 37 (0.18 g) using same
procedure as for the conversion of 17 to 18. IR (mmax): 3420, 2945,
1713, 1683, 1634, 1599 cm−1. 1H NMR: d 7.99 (d, 1H, J = 7.8 Hz,
Ar–H), 7.64 (d, 1H, J = 16 Hz, CH), 7.60 (d, 1H, J = 7.3 Hz,
Ar–H), 7.56 (t, 1H, J = 6.6 Hz, Ar–H), 7.39 (t, 1H, J = 6.6 Hz,
Ar–H), 6.27 (d, 1H, J = 16 Hz, CH), 5.67 (s, 1H, CH), 4.16 (s,
1H, CH), 3.62 (s, 3H, CH3), 3.46 (s, 3H, CH3), 3.34 (s, 3H, CH3),
2.89 (d, 1H, J = 18 Hz, CH2), 2.58 (d, 1H, J = 18 Hz, CH2),
1.95 (s, 3H, CH3), 1.11 (s, 3H, CH3). 13C NMR: d 196.7, 166.3,
149.3, 145.6, 138.5, 134.6, 131.2, 128.9, 127.9, 127.0, 126.4, 118.3,
110.6, 77.1, 59.5, 57.9, 51.1, 48.5, 44.5, 21.2, 16.2. HRMS (m/z)
C21H26O6 requires: 374.1729; found: 374.1736.
(3ꢀZ)-(1R,2S/1S,2R)-2-Dimethoxymethyl-1-[5ꢀ -hydroxy-3ꢀ-
methylpent-3ꢀ -en-1ꢀ -ynyl]-2-methyl-1,2,3,4-tetrahydronaphthalen-
1-ol (35) and (3ꢀZ)-(1R,2R/1S,2S)-2-Dimethoxymethyl-1-[5ꢀ -
hydroxy-3ꢀ-methylpent-3ꢀ-en-1ꢀ-ynyl]-2-methyl-1,2,3,4-tetra-hydro-
naphthalen-1-ol (36). The side chain was added to 34 (24.4 g,
15.4 mmol) in the same manner as for compound 13. Column
chromatography using silica gel with hexane–EtOAc (6 : 1 to 4 :
1) afforded two diastereomeric products 35 (Rf = 0.13 hexane–
EtOAc 1 : 1, 18.1 g, 52.8%) and 36 (Rf = 0.17 hexane–EtOAc 1 :
1, 10.6 g, 30.8%), both as light yellow solids. IR (mmax); 3380, 3055,
2940, 2278, 1602 cm−1.
Methyl (2Z,4E)-(1ꢀR,2ꢀS/1ꢀS,2ꢀR)-5-[2ꢀ-formyl-1ꢀ-hydroxy-2ꢀ-
methyl-4ꢀ-oxo-1ꢀ,2ꢀ,3ꢀ,4ꢀ-tetrahydronaphthalen-1ꢀ-yl]-3-methylpenta-
2,4-dienoate (39). The ester 38 (0.250 g, 0.688 mmol) was
dissolved in CHCl3 (8 mL) to which 50% aq. TFA (4 mL) was
then added. The reaction mixture was refluxed for 4 h and then
allowed to cool to rt. The CHCl3 layer was washed with H2O
(25 mL) followed by a saturated solution of NaHCO3 (25 mL)
and then dried over anhydrous Na2SO4. PrepTLC on silica gel
coated glass plate (20 × 20 cm) using 33% ethyl acetate in hexane
yielded aldehyde 39 (0.180 g, 82%). IR (mmax): 3432, 2949, 2845,
1717, 1686, 1636, 1599 cm−1. 1H NMR: d 9.66 (s, 1H, CHO), 8.00
(d, 1H, J = 7.7 Hz, Ar–H), 7.72 (d, 1H, J = 16 Hz, CH),7.59 (m,
2H, 2 × Ar–H), 7.40 (m, 1H, Ar–H), 6.19 (d, 1H, J = 16 Hz,
CH), 5.72 (s, 1H, CH), 3.63 (s, 3H, CH3), 3.09 (d, 1H, J = 18 Hz,
CH), 2.67 (d, 1H, J = 18 Hz, CH), 1.95 (s, 3H, CH3), 1.27 (s, 3H,
CH3). 13C NMR: d 205.0, 194.3, 166.3, 148.7, 145.7, 136.2, 135.0,
130.6, 129.3, 128.6, 127.1, 126.5, 119.1, 54.8, 51.2, 43.4, 21.1,
16.7. HRMS (m/z) C19H20O5 requires: 328.1311; found: 328.1314.
Compound 35. 1H NMR: d 7.76 (m, 1H, Ar–H), 7.17 (m, 2H,
Ar–H), 7.07 (m, 1H, Ar–H), 5.85 (t, 1H, J = 6.8 Hz, CH), 4.53 (s,
1H, CH), 4.27 (d, 2H, J = 6.8 Hz, CH2), 3.56 (s, 6H, OCH3), 2.86
(m, 2H, CH2), 2.23 (m, 1H, CH2), 1.91 (s, 3H, CH3), 1.83 (m, 1H,
CH2), 1.04 (s, 3H, CH3),. 13C NMR: d 144.3, 144.6, 135.5, 133.0,
132.8, 128.5, 127.1, 124.4, 110.3, 89.6, 75.8, 58.6, 58.7, 57.8, 50.5,
29.7, 26.7, 24.9, 22.8, and 17.8. HRMS (m/z) C22H29O6 requires:
389.1969 [M + O2CCH3]−; found: 389.1955.
Compound 36. 1H NMR: d 7.72 (d, 1H, J = 7.8 Hz, Ar–H),
7.19 (t, 1H, J = 7.3 Hz, Ar–H), 7.13 (t, 1H, J = 7.3 Hz, Ar–H),
7.02 (d, 1H, J = 7.7 Hz, Ar–H), 5.77 (t, 1H, J = 6.7 Hz, CH),
4.58 (s, 1H, CH), 4.13 (d, 2H, J = 6.7 Hz, CH2), 3.64 (s, 3H,
CH3), 3.61 (s, 3H, CH3), 2.75–2.87 (m, 2H, CH2), 1.81–1.83 (m,
2H, CH2), 1.80 (s, 3H, CH3), 0.95 (s, 3H, CH3). 13C NMR: d 143.3,
143.3, 135.5, 133.8, 132.8, 129.8, 128.3, 124.4, 111.3, 89.6, 76.8,
58.8, 58.7, 57.6, 51.5, 29.7, 27.7, 24.9, 21.8, and 18.8. HRMS (m/z)
C22H29O6 requires: 389.1969 [M + C2H3O2]−; found: 389.1971.
Methyl (2Z,4E)-(1ꢀR,2ꢀR/1ꢀS,2ꢀS)-5-[1ꢀ-hydroxy-2ꢀ-hydroxy-
methyl-2ꢀ -methyl-4ꢀ -oxo-1ꢀ,2ꢀ,3ꢀ,4ꢀ-tetrahydronaphthalen-1ꢀ-yl]-3-
methylpenta-2,4-dienoate (40). To the aldehyde 39 (0.100 g,
0.305 mmol) in CH3OH (5 mL) was added 0.1 g of NaBH4 and
mixture stirred at rt for 20 min. H2O (5 mL) was then added and
the mixture stirred for a further 30 min. The reaction mixture
was then extracted with ethyl acetate (3 × 25 mL), washed with
saturated NaCl solution and dried over anhydrous Na2SO4.
The solvent was removed in vacuo, leaving a residue of the tri-
hydroxy product (0.077 g), which was carried through to the next
stage without further purification. To a stirred solution of the
trihydroxy intermediate (0.077 g, 0.233 mmol) in reagent grade
acetone (20 mL) was added MnO2 (0.406 g, 4.67 mmol). The
Methyl (2Z,4E)-(1ꢀR,2ꢀS/1ꢀS,2ꢀR)-5-[2ꢀ-dimethoxymethyl-1ꢀ-
hydroxy - 2ꢀ - methyl - 1ꢀ,2ꢀ,3ꢀ,4ꢀ - tetrahydronaphthalen - 1ꢀ - yl] - 3 -
methylpenta - 2,4 - dienoate (37). Allylic alcohol 35 (1.30 g,
3.94 mmol) was converted to ester 37 (0.65 g, 37% over three
steps) in the same manner as the conversion of 15 to 17. IR (mmax):
3434, 2946, 1714, 1631, 1600 cm−1. 1H NMR: d 7.62 (d, 1H, J =
16 Hz, CH), 7.42 (d, 1H, J = 7.3 Hz, Ar–H), 7.16 (m, 3H, Ar–H),
6.32 (d, 1H, J = 16 Hz, CH), 5.66 (s, 1H, CH), 4.23 (s, 1H, CH),
3.64 (s, 3H, CH3), 3.56 (s, 3H, CH3), 3.42 (s, 3H, CH3), 2.86 (m,
2H, CH2), 2.12 (m, 1H, CH), 1.99 (s, 3H, CH3), 1.76 (m, 1H,
CH), 1.02 (s, 3H, CH3),. 13C NMR: d 166.5, 150.1, 142.2, 140.6,
135.1, 128.5, 127.8, 127.1, 126.1, 117.2, 110.8, 77.3, 60.4, 58.7,
51.0, 45.0, 25.8, 25.1, 21.3, 16.2. HRMS (m/z) C21H28O5 requires:
360.1937; found: 360.1935.
R
reaction mixture was stirred at rt for 2 h, filtered over a Celiteꢀ
bed and the filtrate concentrated in vacuo. The resulting residue
was purified by PrepTLC on silica gel coated plate (20 × 20 cm)
using EtOAc–hexane (2 : 3) to give the desired hydroxyester 40
(0.074 g, 74%) over the two steps. IR (mmax): 3477, 3060, 2987,
1
1704, 1686, and 1594 cm−1. H NMR: d 8.02 (dd, 1H, J = 7.8
and 1.2 Hz, Ar–H), 7.87 (d, 1H, J = 16.0 Hz, CH), 7.55 (dt, 1H,
J = 7.3 and 1.3 Hz, Ar–H), 7.50 (d, 1H, J = 7.1 Hz, Ar–H), 7.39
(dt, 1H, J = 7.3 and 1.3 Hz, Ar–H), 6.31 (d, 1H, J = 16.0 Hz,
CH), 5.74 (s, 1H, CH), 3.71 (d, 1H, J = 11.3 Hz, CH2), 3.65
(s, 3H, CH3), 3.57 (d, 1H, J = 11.2 Hz, CH2), 3.08 (d, 1H, J =
18.0 Hz, CH), 2.47 (d, 1H, J = 18.0 Hz, CH), 2.01 (s, 3H, CH3),
1.03 (s, 3H, CH3). 13C NMR: d 197.3, 166.6, 149.7, 145.3, 137.9,
134.6, 130.8, 129.1, 128.4, 127.3, 126.8, 118.1, 79.3, 69.8, 51.2,
Methyl (2Z,4E)-(1ꢀR,2ꢀS/1ꢀS,2ꢀR)-5-[2ꢀ-dimethoxymethyl-1ꢀ-
hydroxy-2ꢀ-methyl-4ꢀ-oxo-1ꢀ,2ꢀ,3ꢀ,4ꢀ-tetrahydronaphthalen-1ꢀ-yl]-3-
methylpenta-2,4-dienoate (38)16. Benzylic oxidation of ester 37
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45.4, 43.8, 21.3, 19.8. HRMS (m/z) C19H22O5 requires: 330.1467;
found: 330.1467.
1H, J = 16 Hz, CH), 5.68 (s, 1H, CH), 4.20 (s, 1H, CH), 3.67
(s, 3H, CH3), 3.59 (s, 3H, CH3), 3.53 (s, 3H, CH3), 2.67 (d, 1H,
J = 18 Hz, CH), 2.62 (d, 1H, J = 18 Hz, CH), 1.91 (s, 3H, CH3),
1.07 (s, 3H, CH3). 13C NMR: d 195.8, 166.3, 149.4, 145.8, 139.3,
134.9, 129.5, 127.5, 127.1, 126.4, 126.0, 118.1, 111.4, 76.7, 60.8,
57.2, 51.1, 48.3, 44.8, 21.3, 13.9. HRMS (m/z) C22H27O8 requires:
419.1711 [M + CHO2]−; found: 419.1718.
(2Z,4E)-(1ꢀS,2ꢀS)-5-[1ꢀ-Hydroxy-2ꢀ-hydroxymethyl-2ꢀ-methyl-
4ꢀ-oxo-1ꢀ,2ꢀ,3ꢀ,4ꢀ-tetrahydronaphthalen-1ꢀ-yl]-3-methylpenta-2,4-
dienoic acid [(+)-10].
Resolving the dihydroxyester 40 by chiral HPLC. Dihydrox-
yester 40 was resolved on a chiral HPLC column (250 × 10 mm,
Whelk-01, Kromasil, Regis Technologies Inc., USA) with 6% IPA
in hexane at 7.5 mL min−1 and UV detection at 262 nm, to afford
(+)-40 [a]2D5 +189 (c 1.57, CHCl3) and (−)-40 [a]2D5 −191 (c 1.43,
CHCl3) with retention times of 20.5 and 24.2 min, respectively.
The resolved esters were hydrolyzed to the corresponding acids
by using esterase from porcine liver (Sigma Cat. No. E-3019) as
follows: Theester (+)-40 (15.7mg, 4.76 × 10−5mmol)was dissolved
in MeOH (10 drops) to which a phosphate buffer at pH 8.0
(1.5 mL) was added. The pH of the mixture was maintained at
8.8 using 1.0 M KOH. The reaction mixture was stirred at rt for
24 h after which all the starting material had disappeared. MeOH
was evaporated in vacuo and reaction mixture acidified with 10%
HCl. The reaction mixture was then extracted with EtOAc, washed
with saturated NaCl, dried over anhydrous Na2SO4 and solvent
evaporated in vacuo to afford the (+)-enantiomer of the acid (+)-
10 (9.2 mg, 61%), as an oil. [a]2D5 +163 (c 0.92, CHCl3). IR (mmax):
3610–2457, 3452, 1684, 1601 cm−1. 1H NMR: d 8.00 (d, 1H, J =
7.7 Hz, Ar–H), 7.82 (d, 1H, J = 16.0 Hz, CH), 7.56 (t, 1H, J =
7.5 Hz, Ar–H), 7.48 (d, 1H, J = 7.2 Hz, Ar–H), 7.40 (t, 1H, J =
7.5 Hz, Ar–H), 6.31 (d, 1H, J = 16.0 Hz, CH), 5.74 (s, 1H, CH),
3.72 (d, 1H, J = 11.2 Hz, CH), 3.55 (d, 1H, J = 11.2 Hz, CH),
3.07 (d, 1H, J = 17.3 Hz, CH), 2.48 (d, 1H, J = 17.3 Hz, CH),
2.04 (s, 3H, CH3), 1.03 (s, 3H, CH3). 13C NMR: d 197.7, 170.1,
151.4, 144.9, 138.4, 134.6, 130.7, 129.3, 128.5, 127.4, 126.8, 118.0,
79.5, 69.6, 45.2, 43.7, 21.6, 19.8. HRMS (m/z) C18H19O5 requires:
315.1237 [M − 1]−; found: 315.1232. The ester (−)-40 (14.3 mg,
4.33 × 10−5 mmol) was treated the same way as above and afforded
(−)-10 (8.6 mg, 63%), as an oil. [a]2D5 −160 (c 1.34, CHCl3).
Diastereomer 36 was transformed and resovled to give (+)-11
and (−)-11 using the same procedure for the conversion of 35 to
(+)-10 and (−)-10.
Methyl (2Z,4E)-(1ꢀR,2ꢀR/1ꢀS,2ꢀS)-5-[2ꢀ-formyl-1ꢀ-hydroxy-2ꢀ-
methyl-4ꢀ-oxo-1ꢀ,2ꢀ,3ꢀ,4ꢀ-tetrahydronaphthalen-1ꢀ-yl]-3-methylpenta-
2,4-dienoate (diastereomer of 39). IR (mmax): 3455, 2930, 1714,
1
1685, 1636, 1600 cm−1. H NMR: d 9.73 (s, 1H, CHO), 8.06 (d,
1H, J = 7.8 Hz, Ar–H), 7.74 (d, 1H, J = 16 Hz, CH), 7.61 (t, 1H,
J = 7.3 Hz, Ar–H), 7.57 (d, 1H, J = 7.1 Hz, Ar–H), 7.45 (t, 1H,
J = 7.3 Hz, Ar–H), 6.29 (d, 1H, J = 16 Hz, CH), 5.72 (s, 1H,
CH), 3.63 (s, 3H, CH3), 2.95 (d, 1H, J = 18 Hz, CH), 2.83 (d, 1H,
J = 18 Hz, CH), 1.93 (s, 3H, CH3), 1.26 (s, 3H, CH3). 13C NMR: d
203.5, 194.9, 166.3, 148.8, 143.9, 136.8, 134.9, 130.5, 129.3, 128.7,
127.0, 126.6, 119.1, 76.5 55.1, 51.2, 42.4, 21.1, 16.7. HRMS (m/z)
C20H21O7 requires: 373.1292 [M + CHO2]−; found: 373.1299.
Methyl (2Z,4E)-(1ꢀR,2ꢀS/1ꢀS,2ꢀR)-5-[1ꢀ-hydroxy-2ꢀ-hydroxy-
methyl-2ꢀ -methyl-4ꢀ -oxo-1ꢀ,2ꢀ,3ꢀ,4ꢀ -tetrahydronaphthalen-1ꢀ -yl]-3-
methylpenta - 2,4 - dienoate (diastereomer of 40). IR (mmax): 3437,
1
2948, 1686, 1633, 1599 cm−1. H NMR: d 7.99 (dd, 1H, J = 7.8
and 1.2 Hz, Ar–H), 7.75 (d, 1H, J = 15.9 Hz, CH), 7.60 (dd, 1H,
J = 7.2 and 1.5 Hz, Ar–H), 7.58 (dt, 1H, J = 7.3 and 1.1 Hz,
Ar–H), 7.36 (dt, 1H, J = 7.3 and 1.1 Hz, Ar–H), 6.32 (d, 1H,
J = 15.9 Hz, CH), 5.68 (s, 1H, CH), 3.89 (d, 1H, J = 10.5 Hz,
CH2), 3.64 (s, 3H, CH3), 3.44 (d, 1H, J = 10.6 Hz, CH2), 2.50
(d, 1H, J = 17.5 Hz, CH), 2.34 (d, 1H, J = 17.5 Hz, CH), 1.92
(s, 3H, CH3), 1.18 (s, 3H, CH3). 13C NMR: d 195.8, 166.8, 150.4,
146.3, 138.8, 134.8, 129.7, 127.7, 127.5, 126.6, 126.3, 117.8, 78.4,
69.8, 51.3, 45.0, 44.0, 21.5, 18.6. HRMS (m/z) C19H21O5 requires:
329.1394 [M − H]−; found: 329.1394.
(2Z,4E)-(1ꢀR,2ꢀS)-5-[1ꢀ -Hydroxy-2ꢀ -hydroxymethyl-2ꢀ -methyl-
4ꢀ -oxo-1ꢀ,2ꢀ,3ꢀ,4ꢀ -tetrahydronaphthalen-1ꢀ -yl]-3-methylpenta-2,4-
dienoic acid [(+)-11]. The diastereomer of 40 was resolved as
described earlier to afford (+)-(1ꢀR,2ꢀS)-ester, [a]D25 +272 (c 0.87,
CHCl3) and (−)-(1ꢀS,2ꢀR)-ester, [a]D −277 (c 1.01, CHCl3). The
retention times were 17.7 and 20.2 min, respectively.
Methyl (2Z,4E)-(1ꢀR,2ꢀR/1ꢀS,2ꢀS)-5-[2ꢀ-dimethoxymethyl-1ꢀ-
hydroxy-2ꢀ-methyl-1ꢀ,2ꢀ,3ꢀ,4ꢀ-tetrahydronaphthalen-1ꢀ-yl]-3-methyl-
penta-2,4-dienoate (diastereomer of 37). IR (mmax): 3476, 2944,
1713, 1632, 1599 cm−1. 1H NMR: d 7.76 (d, 1H, J = 16 Hz, CH),
7.48 (d, 1H, J = 7.5 Hz, Ar–H), 7.03–7.14 (m, 3H, Ar–H), 6.38
(d, 1H, J = 16 Hz, CH), 5.65 (s, 1H, CH), 4.03 (s, 1H, CH), 3.68
(s, 3H, CH3), 3.58 (s, 3H, CH3), 3.48 (s, 3H, CH3), 2.82–2.97 (m,
2H, CH2), 1.92 (s, 3H, CH3), 1.80 (m, 2H, CH2), 1.04 (s, 3H,
CH3). 13C NMR: d 166.5, 150.2, 142.5, 139.3, 133.7, 128.0, 127.9,
126.7, 126.6, 124.4, 117.1, 113.1, 60.7, 57.3, 51.0, 45.7, 27.7, 24.6,
The resolved esters were hydrolyzed using esterase as described
for 40 above. The (+)-(1ꢀR,2ꢀS)-ester (15.0 mg, 4.55 × 10−5 mmol)
afforded the corresponding acid (+)-11 (9.0 mg, 63%), [a]D25
+
256 (c 0.18, CHCl3) while the (−)-(1ꢀS,2ꢀR)-ester (16.0 mg. 4.85 ×
10−5 mmol) yielded the acid (−)-11 (9.4 mg, 61%), [a]2D5 −259 (c
0.10, CHCl3). IR (mmax): 3652–2455, 3400, 1684, 1599 cm−1. H
1
•
21.4, 12.3. C21H28O5Na requires: 383.1828 [M + Na]+ ; found:
NMR: d 8.00 (d, 1H, J = 7.8 Hz, Ar–H), 7.70 (d, 1H, J = 16.0 Hz,
CH), 7.63 (d, 1H, J = 7.8 Hz, Ar–H), 7.60 (t, 1H, J = 7.2 Hz,
Ar–H), 7.40 (t, 1H, J = 7.2 Hz, Ar–H), 6.37 (d, 1H, J = 16.0 Hz,
CH), 5.70 (s, 1H, CH), 3.91 (d, 1H, J = 10.5 Hz, CH), 3.45 (d,
1H, J = 10.5 Hz, CH), 2.53 (d, 1H, J = 17.5 Hz, CH), 2.33 (d,
1H, J = 17.5 Hz, CH), 1.96 (s, 3H, CH3), 1.20 (s, 3H, CH3).
13C NMR: d 196.7, 169.0, 149.7, 146.4, 138.9, 134.7, 129.7, 127.5,
127.0, 126.4, 126.2, 118.7, 78.5, 68.9, 44.9, 43.8, 21.2, 18.4. HRMS
(m/z) C18H19O5 requires: 315.1237 [M − 1]−; found: 315.1242.
383.1833.
Methyl (2Z,4E)-(1ꢀR,2ꢀR/1ꢀS,2ꢀS)-5-[2ꢀ-dimethoxymethyl-1ꢀ-
hydroxy-2ꢀ-methyl-4ꢀ-oxo-1ꢀ,2ꢀ,3ꢀ,4ꢀ-tetrahydronaphthalen-1ꢀ-yl]-3-
methylpenta-2,4-dienoate (diastereomer of 38). IR (mmax): 3465,
1
2946, 1712, 1682, 1632, 1599 cm−1. H NMR: d 7.96 (dd, 1H,
J = 7.9 and 1.2 Hz, Ar–H), 7.84 (d, 1H, J = 16 Hz, CH), 7.67
(dd, 1H, J = 7.3 and 0.7 Hz, Ar–H), 7.57 (dt, 1H, J = 6.6 and
1.2 Hz, Ar–H), 7.34 (dt, 1H, J = 6.7 and 1.2 Hz, Ar–H), 6.33 (d,
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10 C. R. Hampson, M. J. T. Reaney, G. D. Abrams, S. R. Abrams and
L. V. Gusta, Phytochemistry, 1992, 31(8), 2645–2648.
11 R. Zhou, A. J. Cutler, S. J. Ambrose, M. M. Galka, K. M. Nelson,
T. M. Squires, M. K. Loewen, A. S. Jadhav, A. R. S. Ross, D. C. Taylor
and S. R. Abrams, Plant Physiol., 2004, 134(1), 361–369.
12 N. M. Irvine, P. A. Rose, A. J. Cutler, T. M. Squires and S. R. Abrams,
Phytochemistry, 2000, 53(3), 349–355.
13 J. M. Nyangulu, M. M. Galka, A. Jadhav, Y. Gai, C. M. Graham, K. M.
Nelson, A. J. Cutler, D. C. Taylor, G. M. Banowetz and S. R. Abrams,
J. Am. Chem. Soc., 2005, 127, 1662–1664.
Acknowledgements
The authors thank the staff of the PBI Spectroscopy labs, Andrew
Ross, Steve Ambrose and Brock Chatson for their contributions
and Garth Abrams for helpful discussions over the course of
this research. The financial support of NSERC for an NSERC
Strategic Grant STRGP234754 and to Genome Prairie Functional
Genomics of Abiotic Stress (to SRA) is gratefully acknowledged.
The crystallographic analysis and some mass spectrometry was
performed at the Saskatchewan Structural Sciences Centre at the
University of Saskatchewan. This paper is National Research
Council of Canada No. 48,017.
14 M. P. Coogan, R. Haigh, A. Hall, L. D. Harris, D. E. Hibbs, R. L.
Jenkins, C. L. Jones and N. C. O. Tomkinson, Tetrahedron, 2003, 59(37),
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15 H. J. Mayer, N. Rigassi, U. Schwieter and B. C. L. Weedon, Helv. Chim.
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