Synthesis of oxygenated cineole derivatives from cineole: Utility of cytochrome P450cin as an enantioselective catalyst

Article abstract of DOI:10.1016/j.tetasy.2013.02.004

The oxidation of cineole to (1R)-6β-hydroxycineole by cytochrome P450_cin (CYP176A) is utilised as the initial step in the synthesis of a range of compounds, both novel and ones previously known only as racemates. The potential of P450_cin to provide useful, enantiopure building blocks is thus demonstrated. In particular, hydroxylation by this enzyme was used as the initial step in the synthesis of a range of functionalised cineole derivatives that could be used as mechanistic probes to elucidate the effect of substrate structure on the transformations mediated by P450_cin.

Full text of DOI:10.1016/j.tetasy.2013.02.004

Tetrahedron: Asymmetry 24 (2013) 324–333
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Synthesis of oxygenated cineole derivatives from cineole: utility of
cytochrome P450cin as an enantioselective catalyst
⇑
Anthony J. Farlow, Paul V. Bernhardt, James J. De Voss
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 January 2013
Accepted 7 February 2013
The oxidation of cineole to (1R)-6b-hydroxycineole by cytochrome P450cin (CYP176A) is utilised as the
initial step in the synthesis of a range of compounds, both novel and ones previously known only as race-
mates. The potential of P450cin to provide useful, enantiopure building blocks is thus demonstrated. In
particular, hydroxylation by this enzyme was used as the initial step in the synthesis of a range of func-
tionalised cineole derivatives that could be used as mechanistic probes to elucidate the effect of substrate
structure on the transformations mediated by P450cin
.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
that includes two required redox-protein partners, cindoxin and
E. coli flavodoxin reductase, has been developed for the in vitro
analysis of the products of P450cin catalysed oxidation of small
molecules.5 Recently, we have demonstrated that an in vivo
bacterial system can be constructed and utilised to produce
(1R)-6b-hydroxycineole 2 from 1 in gram quantities.6 Cineole 1 is
chemically inert and difficult to functionalise regioselectively
and/or stereoselectively by standard chemical methods. To date
oxidation with aqueous basic potassium permanganate has been
reported to form rac-cineolic acid 3 whilst treatment with chromyl
acetate forms rac-5-ketocineole 4 (Fig. 2). A range of compounds
have been synthesised from these two precursors,7 but neither
transformation is enantioselective. The enantioselective synthesis
of both enantiomers of 6b-hydroxycineole 2 can be achieved from
Cytochromes P450 are a superfamily of oxidative hemoproteins
best known for the hydroxylation of unactivated hydrocarbons.1
Cytochrome P450cin (CYP176A), isolated from the bacterium Citro-
bakter braakii and initially reported in 2002,2 carries out a highly
regio- and enantioselective oxidation of cineole 1 to (1R)-6b-
hydroxycineole 2 (Fig. 1).3
10
5
11
8
3
2O
4
O
HO
1
7
a
-terpineol,8 but the yields are
6
the appropriate enantiomer of
1
2
9
modest and enantiomerically-pure
a
-terpineol is typically
expensive.
Figure 1. Hydroxylation of cineole 1 to 2 mediated by cytochrome P450cin
.
Cytochrome P450cin has been cloned and can be overexpressed
at high levels in Escherichia coli, purified and stored,2 and has thus
proven to be a useful model system for probing the structure and
chemistry of P450s.3a,4 A reconstituted, catalytically active system
O
1
CrO3
(CH3CO)2O
KMnO4
⇑
Corresponding author. Tel.: +61 (7) 3365 3825; fax: +61 (7) 3365 4299.
E-mail address: j.devoss@uq.edu.au (J.J. De Voss).
There are inconsistencies in the nomenclature of cineole derivatives in the
O
O
O
literature. Herein we use
a
modification of the system devised by Carman and
- or b-substituents if
HOOC
Fletcher4 who defined substituents on the carbocyclic ring as
a
HOOC
they were endo- or exo- to the carbocyclic ring, respectively. We apply the IUPAC
priority rules in numbering the carbon atoms of the cineole framework and define the
stereochemistry of the C1 carbon in chiral molecules. The product of P450cin oxidation
of cineole, in accordance with the rules described, is named (1R)-6b-hydroxycineole
2.
3
4
Figure 2. Oxidative functionalisation of cineole
1 by standard chemical
transformations.
0957-4166/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2013.02.004

A. J. Farlow et al. / Tetrahedron: Asymmetry 24 (2013) 324–333
325
We wished to synthesise a range of enantiomerically pure,
substituted keto- and diol derivatives of cineole. These were
intended to be probe substrates with which to investigate a num-
ber of poorly understood processes catalysed by P450s including
oxidative C–C bond cleavage of vicinal diols and Baeyer–Villiger-
like oxidation of carbonyl containing compounds.1,9 In particular,
the absolute and relative stereochemistry of the substituents was
to be an important factor in understanding the mechanism of these
transformations. Thus, we used the in vivo bacterial system we had
previously developed6 for the production of enantiopure 2 from
readily available cineole 1 as an attractive initial step in the syn-
thesis of the desired stereochemically defined derivatives. Herein
we report the synthesis and characterisation of these compounds
and concomitantly demonstrate the utility of our bacterial system
expressing functional P450cin for the production of synthetically
useful quantities of enantiopure building blocks.
Deprotonation of 5 with lithium diisopropylamide (LDA) and
subsequent treatment of the resulting enolate with bromine gave
(1R)-5a-bromo-6-ketocineole 6, previously reported with unde-
fined absolute configuration (Fig. 4).11 Treatment of the enolate
of 5 with iodomethane afforded the previously unreported (1R)-
5a-methyl-6-ketocineole 7 (Fig. 4). The stereochemistry of C5 in
these products was clear from the 1H NMR spectrum as the 5b pro-
ton in both cases exhibited characteristic W-coupling with the 8b
proton. None of the products with a 5b substituent were detected
in these reactions. In contrast, fluorination of the enolate of 5 with
N-fluorobenzyl sulfonamide (NFSI) led to a mixture of (1R)-5a-flu-
oro-6-ketocineole 8 and its 5b-epimer 9, with the formation of the
latter favoured by an excess of base. In the case of 8, the C5Hb–
C8Hb protons did not exhibit a W-coupling and the epimers were
differentiated by the presence in 8 or the absence in 9 of an NOE
interaction between the C5 proton and the C10 methyl group.
The formation of these two epimers at C5 must presumably be
due to the relatively small ionic radius of fluorine allowing forma-
tion of the hindered 5b-epimer 9 because of the reduced steric
interaction between the 5b-substituent and the adjacent methyl
substituent of the bridge.
Both (1S)-6-ketocineole ent-5 and (1R)-6-ketocineole 5 have
previously been reported as intermediates in the biodegradation
of cineole by a number of organisms.12 It was proposed that
(1S)-6-ketocineole ent-5 underwent a biological Baeyer–Villiger-
like oxidation and subsequent rearrangement to yield 5,5-di-
methyl-(4R)-(3-oxobutyl)dihydrofuran-2-one ent-10 as this had
been isolated from the microbial degradation of cineole.12 We
found that simple treatment of 5 with peracid did not yield the de-
sired lactone. However, facile oxidation of 5 to 10 was achieved
using hydrogen peroxide, even in the absence of an acid catalyst
2. Results and discussion
(1R)-6-Ketocineole 5 was readily available from bacterially pro-
duced (1R)-6b-hydroxycineole 2 via Swern oxidation. Oxidation of
2 with chromium based reagents produced ketone 5 contaminated
with trace amounts of lactone 10 vide infra. Enantioselective GC
(b-cyclodextrin CYDEX-B column10) analysis of ketone 5 (Fig. 3) syn-
thesised via this route demonstrated both its enantiomeric purity
and the problems of synthesis from commercial
a-(À)-terpineol
via the procedure reported by Carman and Fletcher;8 the latter yields
a mixture of enantiomers. This ketone 5 was the key intermediate
in the synthesis of a range of substituted ketocineole derivatives.
(Fig. 5). A detailed mechanism for the oxidation of
a-hydroxy ke-
tones and -alkoxy ketones by hydrogen peroxide that differs
a
slightly from the accepted Baeyer–Villiger transformation has been
described before by Ogata et al.13 and we propose that the oxida-
tion of (1R)-6-ketocineole
5 to 5,5-dimethyl-(4S)-(3-oxobu-
tyl)dihydrofuran-2-one 10 proceeds via the same mechanism
(Fig. 5). It was of interest to determine whether lactone 10 formed
spontaneously at low pH in an aqueous medium. It was unclear
whether an in vitro assay with P450cin would yield 10, which
would be easily extracted with an organic solvent, or the presum-
ably water-soluble corresponding hydroxyacid. Basic hydrolysis of
10 was confirmed by NMR analysis. Acidification to pH 1 and
extraction with chloroform resulted in good recovery of lactone
10, indicating its facile formation from the acid at a low pH.
A number of 5,6 dioxygenated derivatives were also synthesised
from ketocineole 5 via (1R)-5,6-diketocineole 11. Although 11 had
Figure 3. Enantioselective GC analysis of (1R)-6-ketocineole
5 derived from
commercial
a-(À)-terpineol (–top) and P450cin oxidation of cineole 1 (— bottom).
O
Swern
O
HO
2
O
5
1. LDA
2. Br2
1. LDA
2. NFSI
1. LDA
2. CH3I
F
O
O
O
O
Br
F
O
O
O
O
6
7
8
9
Figure 4. Base-catalysed addition of selected electrophiles to the enolate of (1R)-6-ketocineole 6.

326
A. J. Farlow et al. / Tetrahedron: Asymmetry 24 (2013) 324–333
O
O
O
H2O2
HO
HOOC
O
O
5
OH
OH-
H2O
HO
O
O
O
HOOC
O
HOOC
O
O
H
10
Figure 5. Hydrogen peroxide-mediated oxidation of (1R)-6-ketocineole 5.
previously been synthesised with an unspecified enantiomeric
purity using selenium dioxide,14 we found that refinements to
the reported work-up were necessary, and here provide extensive
characterisation of the resulting diketone using modern spectro-
scopic techniques. The yellow 1,2-diketone was hygroscopic and,
over time, hydrated to give a white powder. HMBC NMR experi-
ments in D2O revealed an interaction between the C9 methyl
hydrogens and the hydrated but not the unhydrated carbonyl
group, thus demonstrating that hydration occurred at C6 [a similar
NMR investigation of (1R)-6-ketocineole 5 indicated no significant
hydration of the carbonyl occurred in 2H2O]. The melting point of
11 measured herein differed from that which had been previously
reported.14 Great care was taken to keep the diketone free of water,
and differential degrees of hydration may account for the melting
point discrepancies. Similarly, the UV–visible spectrum run in
chloroform herein was bathochromically-shifted by 4–7 nm for
the visible and near-UV absorption maxima and exhibited some-
what stronger molar extinction coefficients compared to the re-
ported UV–visible spectrum in ethanol. Hemiacetal formation at
the electrophilic C6 carbonyl may be insignificant in ethanol and
would thus clearly alter the chromophore (Fig. 6).
As previously observed with 5, oxidation of diketone 11 with
hydrogen peroxide proceeded smoothly and afforded high yields
of enantiomerically pure (1R)-cineolic acid 12, isolated as the di-
methyl ester 13, previously reported as the racemate (Fig. 7).
Derivatives of 6-ketocineole, 5,6-diketocineole and cineolic acid
have been tested for antimicrobial, antiarrhythmic, hypotensive,
anaesthetic and anticancer properties,15 but despite some promis-
ing results these investigations appear not to have been continued.
The enantiopurity of these derivatives was not reported and one
limitation in these previous studies may be that they were con-
ducted with racemic cineole derivatives.
Reduction of diketone 11 with lithium aluminium hydride
afforded all four possible diols 14–17 (Fig. 8). As might be expected
from the preference for formation of the 5a isomers during alkyl-
ation of the enolate derived from 5, the major products observed
here possessed the 5b configuration, presumably due to the methyl
of the bridge directing attack from the least hindered
5b,6b-diol 14 was therefore the major product with much smaller
amounts of the 5b,6 -diol 16 and the 5 ,6b-diol 17 formed. The
,6 -diol 15 was only formed in trace amounts and could not
a face. The
a
a
5a a
be separated from 14 by chromatography. Its presence was con-
firmed only by conversion of a mixture of 14 and 15 to the corre-
sponding acetonides 18 and 19 that were chromatographically
separable; extensive NMR characterisation allowed elucidation of
the structure of 19.
The structures of diols 14–17 and acetonides 18–19 were ini-
tially assigned by 1H NMR spectroscopy. The C5 hydrogen was dis-
tinguishable from the C6 hydrogen by virtue of a 2–4 Hz coupling
O
O
O
SeO2
to the C4 bridgehead hydrogen, whilst the a- or b-configuration at
O
O
C5 and C6 could be established by the presence or absence of W-
couplings to the C8 and C7 b-hydrogens, respectively. In addition,
the magnitude of the vicinal coupling constants between the C5
and C6 hydrogens provided independent confirmation of their rel-
5
11
Figure 6. Synthesis of (1R)-5,6-diketocineole 11 via selenium dioxoide mediated
oxidation of 5.
O
O
O
O
H2O2
CH2N2
MeOOC
MeOOC
13
HOOC
HOOC
O
11
12
Figure 7. Synthesis of cineolic acid dimethyl ester 13 from (1R)-6-ketocineole 5.

A. J. Farlow et al. / Tetrahedron: Asymmetry 24 (2013) 324–333
327
O
O
O
11
LiAlH4
O
HO
O
HO
HO
O
O
HO
HO
+
HO
17
16
OH
(4%)
15
HO
14
(1%)
(54%)
acetone
CuSO4/H2SO4
O
O
O
+
O
O
O
19
18
(27%)
Figure 8. Reduction of (1R)-5,6-diketocineole 11 with lithium aluminium hydride affords all four possible diols 14–17.
Figure 9. ORTEP views (30% ellipsoids) of the diastereomeric diols 14 (left), 16 (centre) and 17 (right).
ative configuration. For example, neither H5 nor H6 displayed any
long range coupling in 14, but in 16 H6 was clearly coupled to H7b
(J 1.8 Hz). Furthermore, in 14 JH5–H6 was 8.6 Hz, typical of a cis cou-
pling constant whilst in 16 and 17 it was 4.2 Hz, indicative of a
trans relative configuration for these two hydrogens. Although
the structural assignments of the four isomers were internally con-
sistent, X-ray crystal structures were subsequently obtained for
diols 14, 16 and 17 (Fig. 9) and confirmed these assignments. The
varying stereochemistry at the 5- and 6-positions leads to different
H-bonding motifs in the solid state. The cis hydroxyl groups in 14
are linked by an intramolecular H-bond and also intermolecular H-
bonds which result in one-dimensional chains parallel to the a axis.
The trans-configurations of 16 and 17 make intramolecular H-
bonding impossible. Compound 16 forms a 3-D network of H-
bonds while compound 17 crystallises in 2-D sheets.
tial Baeyer–Villiger-like reactions catalysed by cytochrome P450cin
as this structural motif is present in a number other systems be-
lieved to undergo P450 catalysed C–C bond cleavage.1,9 Neither di-
rect reduction of diketone 11 nor direct oxidation of diol 14
proceeded with useful selectivity and therefore synthetic routes
employing selective protection of either the C5 or C6 functionality
in 11 or 14 were explored.
Initially, selective monoprotection of diketone 11 was investi-
gated (Fig. 10). It was anticipated that subsequent reduction and
hydrolysis of the acetal would yield the desired a-hydroxy ketone
20. This route was later abandoned because the conditions re-
quired to hydrolyse the acetal also cleaved the ether bridge of cin-
eole. Nonetheless, the ease with which either carbonyl group could
be selectively protected simply by altering the reaction tempera-
ture was intriguing. Considering the ease with which diketone 11
formed a hydrate (vide supra), it was expected that it would read-
ily undergo acetal formation with ethylene glycol. However,
The
a-hydroxycarbonyl present in (1R)-5b-hydroxy-6-ketocine-
ole 20 (Fig. 10) appeared to be a useful probe for exploring poten-

328
A. J. Farlow et al. / Tetrahedron: Asymmetry 24 (2013) 324–333
O
O
O
11
ethylene glycol, CuSO4/H2SO4
reflux (bp 197 oC)
90 min
ethylene glycol, CuSO4/H2SO4
toluene, reflux (bp 110 oC)
12 h
O
O
O
O
O
O
O
22
O
21
1. [H]
2. Deprotection
HO
O
O
20
Figure 10. Selective ketal formation from 5,6-diketocineole 11 gave either 21 or 22 depending on the reaction conditions.
Figure 11. ORTEP views (30% probability ellipsoids) of ketals 21 (left) and 22 (right).
although monoprotection proceeded very slowly at room temper-
ature, the reaction gave smoothly a single monoacetal when water
was removed as an azeotrope with toluene. X-ray crystallography
demonstrated that the monoprotected diketone formed under
these conditions was the C5 ketal 21 (Fig. 11). When the reaction
was carried out in refluxing ethylene glycol (bp 197 °C), only trace
quantities of the C5 acetal were produced and the major product
isolated was the C6 ketal 22, the structure of which was also deter-
mined by X-ray crystallography (Fig. 11). Unlike the diol analogues,
there is an absence of H-bond donors in these molecules, and in the
structure of 22, disorder in the ketal ring is seen in one of the two
crystallographically independent molecules. Presumably the ob-
served selectivity of monoprotection is a kinetically favoured iso-
mer 21 forming at lower temperature and a thermodynamically
favoured one 22 predominating at a higher temperature. This is
supported by simple MM2 calculations (Chem3D) which indicate
that 22 is indeed more stable than 21, although the molecular ori-
gins of this are not clear. It can be speculated that the interaction of
TBDMS-O
HO
HO
HO
TBDMS-Cl
NaH
O
O
23
14
Swern oxidation
(2 steps, 59%)
HO
TBDMS-O
O
TBAF
0oC
O
O
(41%)
O
20
24
Figure 12. Synthesis of (1R)-5b-hydroxy-6-ketocineole 20 from diol 14 via
the methyl substituent of the bridge and the C5
a
substituent dis-
selective protection of the C5 alcohol.

A. J. Farlow et al. / Tetrahedron: Asymmetry 24 (2013) 324–333
329
cussed above destabilises 21 relative to 22; this same interaction
might promote the formation of an oxonium ion from an interme-
diate hemiacetal at C5 and thus kinetically promote acetal forma-
tion at this position.
In addition, the cineole derivatives, thus synthesised will be used
to probe the capacity of P450cin to carry out atypical, P450-medi-
ated oxidative transformations such as Baeyer–Villiger-like oxida-
tions and the C–C bond cleavage of vicinal diols.
In order to synthesise the desired (1R)-5b-hydroxy-6-ketocine-
ole 20 from (1R)-5b,6b-dihydroxycineole 14, a protecting group
was needed that was not only selective for the C5 hydroxy group,
but could also be removed under mild conditions so as to prevent
4. Experimental
4.1. General
isomerisation of the
a-hydroxyketone formed. Several protecting
groups including a benzyl ether, a tetrahydropyran and an acetate
were investigated but the TBDMS ether proved to be the most sat-
isfactory (Fig. 12). The selective formation of the C5 TBDMS ether
was achieved using TBDMS–Cl and sodium hydride in THF. Subse-
quent Swern oxidation afforded the desired protected hydroxy ke-
tone 24 as a single isomer in 59% yield over two steps. Treatment of
24 with TBAF at 0 °C afforded the target compound 20 in reason-
able yield and satisfactory purity for subsequent enzyme assays.
1H NMR revealed only a coupling between the C5 hydrogen and
the C4 hydrogen and no W-coupling to the C-7 hydrogen as ex-
pected for the 5b isomer. However, cognisant of the absence of a
W-coupling in fluorinated ketocineole 8, presumably due to the
strongly electron withdrawing nature of the substituent, we con-
firmed the stereochemistry of 20 via extensive NMR (HSQC, HMBC,
NMR experiments were performed on Bruker Avance 300 MHz,
Oxford Cryogenic Systems 400 MHz, or Bruker Avance 500 MHz
instruments. Unless otherwise specified, NMR samples were dis-
solved in CDCl3. Melting points are corrected to benzoic acid (mp
122 °C). Low-resolution EI mass spectra (70 eV) were carried out
on Shimadzu QP 5000 and QP5050 GC–MS instruments employing
Zebron ZB5-MS columns. High-resolution mass spectra were per-
formed on a Finnigan MAT 900XL Trap mass spectrometer (EI) or
a Bruker MicrOTOF-Q mass spectrometer (ESI) by Mr Graham Mac-
Farlane (UQ mass spectral service). Microanalyses were performed
by Mr. George Blazak (UQ microanalytical service) on a Carlo Erba
C/H/N/S elemental analyser. Optical rotations were performed on a
Perkin–Elmer 241MC Polarimeter and are reported in cm2 gÀ1
.
NOESY) analysis. For example, C5aH was observed to interact by
4.2. (1R)-6-Ketocineole 5
NOESY with the C4 bridgehead hydrogen but not with any methyl
group. The facile isomerisation of 20 (Fig. 13) to other hydroxyke-
tones was indicated by the fact that a sample would exhibit multi-
ple peaks with similar MS fragmentation patterns during GC–MS
analysis, even though NMR spectroscopy indicated the presence
of only one isomer. The ratios of these GC–MS peaks varied with
the temperature of the injector port, which is consistent with ther-
mal enolisation and isomerisation.
DMSO (1.0 ml, 15 mmol) was added to a solution of oxalyl chlo-
ride (0.77 ml, 8.8 mmol) stirring in dichloromethane (30 ml) at
À78 °C under nitrogen. (1R)-6b-Hydroxycineole 23 (1.00 g,
5.9 mmol) was added to this solution. After 30 min, the tempera-
ture of the reaction mixture was raised to 0 °C. The mixture was
again cooled to À78 °C and triethylamine (6 ml, 43 mmol) was
added. Hexane (40 ml) was added and the solution was washed
with water (2 Â 30 ml). The organic extract was dried over MgSO4
and concentrated in vacuo. Flash chromatography of the remaining
residue gave 0.89 g (90%) of the title compound (RF = 0.65 in 1:5
EtAc/hexane). The melting point and NMR in CDCl3 spectra were
HO
consistent with the literature.8,12,16
[
a]
D = À68.4 (c 0.3, ethanol)
HO
O
O
(enantiomer reported16 +70.2). The extent of ketone hydration
was explored by NMR in D2O; no hydrate (d 90–110) could be
observed within the limits of detection. 1H NMR (400 MHz, D2O)
d 2.72 (dm, 1H, J 19.9, CHCO), 2.24 (dd, 1H, J 19.9 and 2.4, CHOH),
2.14–1.89 (3H), 1.58 (m, 1H), 1.44 (m, 1H), 1.28 (s, 3H, CH3), 1.10 (s,
3H, CH3), 0.98 (s, 3H, CH3). 13C NMR (100 MHz, D2O) d 215.3 (C@O),
76.5, 76.4, 40.4, 35.9, 27.8, 27.5, 26.8, 20.6, 18.4. m/z (%) 43 (100),
55 (16), 67 (17), 69 (9.1), 82 (38), 111 (4.1), 140 (4.2), 168
(M+Å, 1.7).
H
O
HO
O
20
H
O
O
O
O
HO
O
HO
4.3. (1R)-5a-Bromo-6-ketocineole 6
OH
(1R)-6-Ketocineole 5 (0.10 g, 0.60 mol) was added to 5 equiv of
LDA in dry THF (3 ml) at À78 °C. Two equivalents of bromine were
then added as a solution in dry THF, and the reaction mixture was
allowed to warm to room temperature. After a further 2 h, the
reaction was quenched by the addition of water (5 ml) and ex-
tracted with ether (2 Â 10 ml). The ether extracts were dried over
MgSO4 and the solvent removed by rotary evaporation. Column
chromatography afforded the title compound (0.086 g, 58%);
RF = 0.63 in 1:5 EtAc/hexane. mp 90–91 °C (Lit.11 mp 94–95 °C).
Figure 13. Epimerisation of (1R)-5b-hydroxy-6-ketocineole 20 presumably occurs
via facile formation of the enol.
3. Conclusion
Using the in vivo hydroxylation of cineole 1 to (1R)-6b-hydroxy-
cineole 2 catalysed by cytochrome P450cin expressed in E. coli as
the preliminary step, a number of novel, enantiomerically pure
cineole derivatives have been synthesised. All of the synthetic
routes utilised (1R)-6-ketocineole 5 as the key intermediate and
a number of 5,6-dioxygenated cineoles were synthesised and char-
acterised by X-ray crystallography. Herein we have demonstrated
the utility of cytochrome P450cin as an enantioselective catalyst
in providing enantiopure starting materials for further synthesis.
[
a
]
D = À15.7 (c 1.0, ethanol). 1H NMR (200 MHz) d 4.95 (dd, 1H, J
2.7 and 1.7, CBr), 2.16 (m, 1H), 1.98 (m, 3H), 1.64 (m, 1H), 1.43
(s, 3H, CH3), 1.30 (s, 3H, CH3), 1.18 (s, 3H, CH3). This is consistent
with the literature for 5a-bromo-6-ketocineole (stereochemistry
undefined).11 13C NMR (50 MHz) d 214.0, 76.3, 75.0, 50.4, 45.2,
31.0, 29.2, 27.7, 19.7, 16.8. m/z (%) 43 (100), 59 (5.3), 81 (8.2), 95
(3.0), 121 (6.1), 139 (4.8), 167 (M+Å–Br, 5.3).

330
A. J. Farlow et al. / Tetrahedron: Asymmetry 24 (2013) 324–333
4.4. (1R)-5
a-Methyl-6-ketocineole 7
4.8. (1R)-5,6-Diketocineole 11
(1R)-6-Ketocineole 5 (0.100 g, 0.60 mmol) in THF (1.0 ml) was
added to a solution of LDA (2.5 equiv) in THF (5 ml) at À78 °C.
Methyl iodide (0.2 ml, 3.2 mmol) was then added and the reaction
mixture was allowed to warm to room temperature. After a further
5 h, the reaction mixture was washed with brine (2 ml), dried over
Na2SO4 and concentrated in vacuo. The residue was purified by col-
umn chromatography (SiO2 1:5 ethyl acetate:hexane) to give the
At first, (1R)-6-ketocineole 5 (4.0 g, 0.024 mol) and selenium
dioxide (CAUTION!) (4.5 g, 0.041 mol) in acetic acid (40 ml) were
heated at reflux until the reaction was complete (monitored by
TLC; approx. 40 h). Saturated sodium carbonate was added slowly
until effervescence ceased and the resulting aqueous mixture was
extracted with ethyl acetate (6 Â 50 ml). The combined extracts
were washed with saturated sodium carbonate (150 ml) and dried
over magnesium sulfate. After removal of the solvent in vacuo, the
residue was purified by column chromatography to yield the title
compound (3.0 g, 67%) as a yellow solid, which turned white on
standing or upon the addition of water (hydration). RF = 0.32 in
1:2 EtAc/hexane. Mp 80 °C (Lit.14 65–68 °C). IR: v = 2978, 1753,
title compound as an oil (84 mg, 76%). [
a
]
D = À22.0 (c 0.36, etha-
nol). IR: v = 2972, 2932, 1731. 1H NMR (500 MHz) d 2.84 (tq, 1H,
J 1.5 and 5.8, C5), 1.97 (m, 2H), 1.61 (m, 3H), 1.39 (s, 3H, CH3),
1.26 (s, 3H, CH3), 1.17 (d, 3H, J 5.8, O@CCHCH3), 1.11 (s, 3H, CH3).
13C NMR (125 MHz) d 214.7 (C6), 76.2, 74.9, 42.5, 41.7, 30.0,
29.0, 27.6, 18.9, 16.0, 13.0. m/z (%) 43 (100), 55 (28), 69 (21), 81
(22), 83 (16), 96 (32), 125 (17), 154 (8.7), 182 (M+Å, 1.4). [Found:
C 72.4, H 10.0. C11H18O2 requires C 72.5, H 10.0.].
1735 (Lit.14 v = 1751). [
a
]
D = À14.5 (c 1.0, chloroform). 1H NMR
(300 MHz) d 2.68 (m, 1H, CH), 2.16–2.38 (m, 2H), 1.82–1.89 (m,
2H), 1.43 (s, 3H, CH3), 1.27 (s, 3H, CH3), 1.22 (s, 3H, CH3). 13C NMR
(75 MHz) d 196.2, 191.9, 78.4, 74.2, 52.9, 30.2, 27.5, 27.0, 18.7,
17.3. 1H NMR (300 MHz, D2O) d 2.37 (dd, 1H, J 4.1 and 1.5, methine)
2.23 (dddd, 1H, J 13.7, 10.4, 4.1 and 3.4, C8), 1.91 (m, 2H), 1.77 (m,
1H), 1.33 (s, 3H, CH3), 1.15 (s, 3H, CH3), 1.12 (s, 3H, CH3). 13C NMR
(75 MHz, D2O) d 214.6 (C@O, C5), 92.0 (OCO, C6), 79.2 (C1), 75.8
(C3), 51.8 (C4), 29.8 (C10/11), 27.2 (C7), 26.8 (C10/11), 18.3(C9),
4.5. (1R)-5a-Fluoro-6-ketocineole 8 and (1R)-5b-fluoro-6-keto-
cineole 9
The ratios of the two fluorinated ketocineoles 8 and 9 were
dependent on the excess of LDA used; reported here is the proce-
dure optimised for (1R)-5
a
-fluoro-6-ketocineole 8. (1R)-6-Keto-
17.7 (C8). UV–Vis (CHCl3) k 463 nm (
e
= 44 MÀ1cmÀ1), 316 nm
= 230 MÀ1 cmÀ1): (H2O)
304
= 25 MÀ1 cmÀ1). Literature14 UV–Vis (EtOH)
= 34 MÀ1 cmÀ1), = 47 MÀ1 cmÀ1),
316 nm
cineole 5 (0.234 g, 1.39 mmol) in THF (5 ml) was added to a
solution of LDA (1.81 mmol; 1.3 equiv) in THF (10 ml) under a
nitrogen atmosphere at -78 °C. The solution was stirred for
10 min, after which N-fluorobenzenesulfonimide (0.46 g,
1.46 mmol) in THF (5 ml) was added. The reaction flask was al-
lowed to warm to room temperature over a period of 2 h and the
reaction was quenched with brine (20 ml). The aqueous layer
was extracted with a further 10 ml of ether and then combined
with the THF fraction and dried over MgSO4. A copious amount
of a gum-like residue containing benzenesulfonimide remained
after the solvent was removed by rotary evaporation. This residue
was extracted with ether (2 Â 20 ml), a solvent in which the ben-
zenesulfonimide was found to be only sparingly soluble, and the
extract was in turn evaporated to dryness. The residue was col-
umned first with EtAc/hexane (1:10), then with EtAc/benzene
(1:10) in order to fully separate the two fluorinated ketocineoles
and unreacted 6-ketocineole 5. NMR assignments of both products
were made with the assistance of HMBC and HSQC.
(e
(e
(e
(e
= 62 MÀ1 cmÀ1), 250 nm
(
e
k
k
470 nm
320 nm
(
e
= 51 MÀ1 cmÀ1). m/z (%) 43 (100), 69 (47.9), 83 (14.5), 108
(23.8), 111 (19.7), 126 (27.4), 154 (1.1), 182 (M+Å, 0.1). [Found:
M+Å = 182.0944 by EI-HRMS. C10H14O3 requires M+Å = 182.0943.]
4.9. 5,5-Dimethyl-(4S)-(3-oxobutyl)dihydrofuran-2-one 10
(1R)-6-Ketocineole was oxidised to lactone 10 by dissolving ke-
tone 5 (200 mg, 1.2 mmol) in a 50% ethanol:water mixture (10 ml)
and adding an excess (>10 equiv) of hydrogen peroxide. After 4 h,
excess hydrogen peroxide was destroyed by the addition of man-
ganese dioxide (1 mg). The solution was diluted with water
(30 ml) and extracted with ethyl acetate (2 Â 20 ml). The com-
bined extracts were dried (MgSO4) and the solvent removed in va-
cuo to give the title compound as a clear oil (190 mg, 83%, >95%
purity by NMR). Recrystallisation from hexane gave the title com-
pound as a white solid. mp 46 °C (lit.16 mp 45 °C). [
a]D = +63.1 (c
4.6. (1R)-5
a
-Fluoro-6-ketocineole 8
0.3, CHCl3) (Lit.16 À58.6 for the enantiomer). 1H NMR (400 MHz)
d 2.56 (dd, J 16.7 and 7.6, 1H, CH2C@O), 2.41 (m, 1H), 2.25 (dd, J
16.7 and 11.6, 1H, CH2C@O), 2.18 (m, 2H), 2.14 (s, 3H, CH3), 1.78
(dddd, 1H, J 13.8, 8.9, 6.6 and 3.8), 1.51 (m, 1H), 1.43 (s, 3H,
CH3), 1.25 (s, 3H, CH3). 13C NMR (100 MHz) d 207.3 (C@O), 175.3
(CO–O), 86.7 (C–O), 45.1, 41.8, 34.7, 30.0, 27.3, 23.2, 21.8. m/z (%)
43 (100), 55 (10), 59 (2.9), 67 (3.0), 81 (2.7), 98 (3.9), 111 (2.8),
143 (1.2), 151 (1.1), 166 (M+Å À18, 1.4).
Yield = 0.088 g (34%). Mp 44–45 °C. [a]D = +44.0 (c 1.0, ethanol).
IR: v = 2980, 2936, 1749. 1H NMR (500 MHz) d 4.74 (dd, 1H, J 49
and 2.4, CHF), 2.27 (m, 2H), 1.98 (m, 1H), 1.62 (m, 2H), 1.35 (s,
3H, CH3CCH3), 1.28 (d, 3H, J 3.5, CH3CCH3), 1.18 (s, 3H, CH3). 13C
NMR (125 MHz) d 203.6 (d, J 9.3, C6), 91.3 (d, J 202, C5), 77.2
(C1), 74.0 (d, J 4.4, C3), 42.4 (d, J 14, C4), 29.4 (d, J 5.4, C10/11),
28.9 (C10/11), 26.6 (C7), 19.7 (d, J 9.4 Hz, C8), 18.9 (C9). m/z (%)
43 (100), 59 (14.2), 67 (4.6), 95 (5.7), 143 (6.3), 158 (2.3). [Found:
C 64.6, H 8.0. C10H15O2F requires C 64.5, H 8.1.]
Lactone 10 (36.8 mg, 0.18 mmol) was dissolved in a mixture of
methanolic sodium hydroxide (1 ml, 0.191 M; 0.19 mmol) and THF
(2 ml). The mixture was stirred at room temperature for 72 h, after
which the solvent was evaporated under nitrogen. The residue was
taken up in D2O and analysed by NMR using dioxane as a standard.
1H NMR (200 MHz, D2O) d 2.49 (m, 1H), 2.24 (m, 1H), 2.06 (s, 3H,
COCH3), 1.87 (m, 1H), 1.70 (m, 2H), 1.22 (m, 2H), 1.05 (6H, s,
2 Â CH3). 13C NMR (50 MHz, D2O) d 205.8 (COCH3), 183.3 (COONa),
74.3 (C–O), 46.3, 42.8, 39.5, 30.0, 26.6, 26.3, 25.1.
The aqueous sodium salt of the lactone formed a cloudy precipi-
tate upon acidification with HCl. The acidified solution was
extracted with CDCl3 (1 ml). 1H NMR analysis of both the D2O and
CDCl3 fractions indicated that lactone 10 had partitioned completely
into the organic solvent as the five-membered lactone. Drying and
evaporation of the CDCl3 returned 22 mg (67%) of the lactone.
4.7. (1R)-5b-Fluoro-6-ketocineole 9
Viscous oil, yield = 0.029 g (12%). [
a]
D = À10.1 (c 1.0, ethanol).
1H NMR (400 MHz) d 5.19 (dd, 1H, J 39 and 2.5, CHF), 2.26 (dq, J
6.0 and 2.5, 1H, methine), 2.07 (m, 1H), 1.92 (app. tm, 2H), 1.71
(m, 1H), 1.42 (s, 3H, CH3CCH3), 1.32 (s, 3H, CH3CCH3), 1.18 (s, 3H,
CH3). 13C NMR (100 MHz) d 206.5 (J 10.6, C6), 89.5 (d, J 188, C5),
77.3 (C1), 74.1 (d, J 12.3, C3), 42.1 (d, J 21, C4), 30.7 (C7), 28.8
(C11), 27.7 (C10), 19.0 (C9), 14.8 (d, J 4.4, C8). m/z (%) 43 (100),
59 (17.7), 81 (10.0), 85 (12.0), 95 (22.1), 100 (12.4), 143 (22.5),
158 (9.6).

A. J. Farlow et al. / Tetrahedron: Asymmetry 24 (2013) 324–333
331
4.10. Dimethyl ester of (1R)-cineolic acid 13
4.2 and 1.8, C6 CHO), 2.40–1.95 (m, 1H), 1.73 (br s, 2H, OH),
1.60–1.72 (m, 2H), 1.38–1.48 (m, 2H), 1.37 (s, 3H, CH3), 1.25 (s,
3H, CH3), 1.12 (s, 3H, CH3). 13C NMR (75 MHz) d 80.4, 79.9, 73.3,
73.2, 42.0, 30.9, 30.0, 24.4, 23.2, 21.5. m/z (%) 43 (100), 69 (44.9),
71 (42.8), 93 (27.8), 108 (46.8), 109 (43.6), 128 (27.2), 186 (M+Å,
0.15). [Found: C 64.4, H 9.8. C10H18O3 requires C 64.5, H 9.7.]
The title compound was synthesised by dissolving an enantio-
merically pure sample of (1R)-5,6-diketocineole (0.20 g, 1.1 mmol)
in a 50% ethanol:water mixture (10 ml) and adding an excess
amount (10 equiv) of hydrogen peroxide. After 24 h, the excess
hydrogen peroxide was destroyed by the addition of manganese
dioxide (1 mg). The volume of the mixture was reduced to approx-
imately 5 ml by rotary evaporation. Next, THF (2 ml) was added
and diazomethane (CAUTION!) (approx. 8 M in ether) was added
portionwise until a yellow end-point had been reached, where-
upon the ether and aqueous layer were separated and the organic
layer dried over magnesium sulfate. Filtration and evaporation of
the organic layer yielded the title compound as an oil in essentially
quantitative yield. 1H NMR was in good agreement with that pre-
4.15. (1R)-5a,6b-Dihydroxycineole 17
This product was further purified by recrystallisation from hex-
ane. RF = 0.38 in ethyl acetate, yield = 0.019 g (1%). Mp 114–115 °C.
[a
]
D = À33.6 (c 1.0, ethanol). IR: v = 2477, 3325, 2936. 1H NMR
(300 MHz) d 4.20 (m, 1H, C5 CHO), 3.24 (d, 1H, J 4.2, C6 CHO),
2.05 (m, 1H, OH), 1.91 (m, 1H), 1.80–1.59 (m, 2H), 1.48 (m, 1H),
1.29 (s, 3H, CH3), 1.24 (s, 3H, CH3), 1.09 (s, 3H, CH3). 13C NMR
(75 MHz) d 80.3, 79.8, 73.4, 73.3, 41.9, 30.9, 30.0, 24.4, 23.2, 21.5.
m/z (%) 43 (100), 69 (53.2), 71 (50.1), 93 (30.3), 108 (55.0), 109
(49.1), 126 (23.9), 128 (19.2), 186 (M+Å, 0.57). [Found: C 64.3, H
9.6. C10H18O3 requires C 64.5, H 9.7.]
viously reported for the racemate.17
[
a]
D = À5.7 (c 1.0, ethanol).
1H NMR (400 MHz) d 3.68 (d, 3H, J 1.1, OCH3) 3.61 (d, 3H, J 1.2,
OCH3), 2.40 (m, 2H), 2.04 (qd, 1H, J 13.7 and 3.7), 1.70 (m, 1H),
1.31 (s, 3H, CH3), 1.30 (s, 3H, CH3), 1.27 (tdd, 1H, J 13.7, 4.4 and
0.9), 1.02 (s, 3H, CH3). 13C NMR (100 MHz) d 175.8, 173.6, 74.8,
74.1, 51.8, 51.5, 51.4, 32.3, 30.8, 29.3, 20.7, 20.6. m/z (%) 43
(100), 55 (25.8), 59 (14.1), 83 (10.1), 107 (25.9), 125 (13.8), 153
(75.6), 185 (M+Å À59, 39.3).
4.16. (1R)-5a,6a-Dihydroxycineole acetonide 18 and (1R)-5b,6b-
dihydroxycineole acetonide 19
Fractions containing a mixture of diols 14 and 15 (1.01 g,
5.43 mmol) were dissolved in acetone (10 ml) and stirred in the
presence of sulfuric acid (0.1 g) and copper(II) sulfate (0.1 g). After
2 h, saturated sodium hydrogen carbonate solution (10 ml) was
added and the mixture was extracted with ethyl acetate
(2 Â 20 ml). The combined organic extracts were dried over mag-
nesium sulfate and evaporated. The mixture was purified by col-
umn chromatography to give acetonides 18 (0.281 g, 23%) and 19
(0.006 g, 0.6%), along with unreacted dihydroxycineole 14
(0.721 g, 72%).
4.11. Reduction of (1R)-5,6-diketocineole 11 with lithium
aluminium hydride
(1R)-5,6-Diketocineole 11 (1.80 g, 9.97 mmol) was dissolved in
tetrahydrofuran (40 ml) and cooled in an ice bath. Lithium alumin-
ium hydride (0.4 g, 10 mmol) was added and the mixture was stir-
red for 2 h. The reaction mixture was raised to room temperature
and stirred for another 2 h, after which excess lithium aluminium
hydride was quenched dropwise with cold water. Sulfuric acid
(1 M) was added until the aluminium salts were fully dissolved.
The tetrahydrofuran was removed and the aqueous solution was
extracted with ether (2 Â 40 ml). The combined organic extracts
were dried over magnesium sulfate and concentrated in vacuo.
The residue was fractionated by column chromatography.
4.17. (1R)-5
a,6a-Dihydroxycineole acetonide 18
RF = 0.32 in 1:5 ethyl acetate/hexane, mp = 74 °C. [a]D = +9.1 (c
1.0, ethanol). IR: v = 2969, 2928, 2877. 1H NMR (300 MHz) d 4.26
(dd, 1H, J 8.6 and 3.6, C5 CHO), 3.78 (d, 1H, J 8.6 C6 CHO), 2.01
(dddd, 1H, J 14, 11, 4.8 and 2.0,), 1.81 (m, 1H, methine), 1.75
(ddd, 1H, J 14, 11, and 7,), 1.57 (s, 3H, CH3), 1.43 (s, 3H, CH3),
1.35 (ddd, 1H, J 14, 11 and 2.0), 1.33 (s, 3H, CH3), 1.23 (m, 1H),
1.21 (s, 3H, CH3), 1.16 (s, 3H, CH3). 13C NMR (75 MHz) d 108.5 (ace-
tal), 77.3, 76.5, 72.7, 70.9, 35.8, 31.1, 29.7, 28.6, 25.3, 23.6, 22.8,
19.7. m/z (%) 41 (19.0), 43 (100), 55 (6.5), 59 (6.0), 85 (32.7), 100
(53.9), 110 (6.8), 153 (1.0), 211 (4.1), 226 (0.8). [Found: C 68.9, H
9.9. C13H22O3 requires C 68.9, H 9.8.]
4.12. (1R)-5b,6b-Dihydroxycineole 14
Yield = 1.01 g (54%). RF = 0.34 in 1:1 ethyl acetate/hexane.
Mp = 120–121 °C. IR: v = 3436, 3267, 2972, 2930, 2883. 1H NMR
(300 MHz) d 3.91 (dd, 1H, J 8.6 and 2.1, C5-CHO) 3.56 (d, 1H, J
8.6, C6-CHO), 2.93 (br s, 2H, OH), 1.95 (ddt, 1H, J 14.6, 11.8 and
5.7), 1.77 (ddd, 1H, J 14.6 Hz, 11.8 and 5.7), 1.61 (dt, 1H, J 4.0
and 2.1), 1.46 (m, 1H), 1.35 (s, 3H, CH3), 1.28 (tm, 1H, J 12.7 and
4.0), 1.20 (s, 3H, CH3), 1.10 (s, 3H, CH3). 13C NMR (75 MHz) d
73.6, 72.0, 70.6, 70.2, 38.7, 31.0, 29.9, 28.1, 23.5, 20.5. m/z (%) 43
(100), 69 (69.7), 71 (65.4), 93 (39.9), 108 (76.6), 109 (71.8), 111
(39.2), 128 (52.4), 186 (M+Å, 0.38) [Found: C 64.2, H 9.8. C10H18O3
requires C 64.5, H 9.7.]
4.18. (1R)-5b,6b-Dihydroxycineole acetonide 19
Viscous oil, RF = 0.46 in 1:5 ethyl acetate/hexane. [
a
]
D = À45.9 (c
0.1, dichloromethane). 1H NMR (400 MHz) d 4.58 (ddd, 1H, J 8.1,
4.0 and 1.4, C5 CHO), 3.87 (dd, 1H, J 8.1 and 1.6, C6 CHO), 1.93
(dddd, 1H, J 14.0, 11.5, 6.0 and 2.1), 1.75 (td, 1H, J 4.0 and 2.1,
methine), 1.71 (ddd, 1H, J 14.0, 11.5 and 3.0), 1.61 (m, 1H), 1.49
(s, 3H, CH3), 1.38 (dddd, 1H, J 14.0, 11.5, 6.5 and 1.6), 1.35 (s, 3H,
CH3), 1.31 (s, 3H, CH3), 1.21 (s, 3H, CH3), 1.10 (s, 3H, CH3). 13C
NMR (100 MHz) d 108.7 (acetal), 77.3, 73.0, 72.7, 72.6, 37.9, 29.2,
28.7, 25.9, 24.8, 24.6, 24.3, 13.7. m/z (%) 43 (100), 55 (9.1), 59
(7.9), 85 (45.1), 100 (79.5), 123 (6.8), 168 (2.6), 211 (3.4), 226
(M+Å, 1.1).
4.13. (1R)-5a,6a-Dihydroxycineole 15
This eluent appeared as a trace impurity that co-eluted with
diol 14 and was identified by GCMS and by later isolation of 19
(vide infra). m/z (%) 43 (100), 55 (24.4), 69 (57.0), 71 (51.0), 83
(20.2), 85 (18.5), 93 (26.2), 108 (61.7), 109 (63.3), 126 (25.6), 128
(23.9).
4.14. (1R)-5b,6a-Dihydroxycineole 16
4.19. (1R)-5-(1,3-Dioxolan-2-yl)-6-ketocineole 21
RF = 0.52 in ethyl acetate. Yield = 0.075 mg (4%). Mp 150 °C.
D = À5.9 (c 1.0, ethanol). IR: v = 3401, 3323, 2971. 1H NMR
[
a
]
(1R)-5,6-Diketocineole 11 (0.60 g, 3.3 mmol), p-toluenesulfonic
acid (10 mg) and ethylene glycol (3 ml) were combined and al-
(300 MHz) d 3.81 (dd, 1H, J 4.2 and 2.1, C5 CHO), 3.70 (dd, 1H J

332
A. J. Farlow et al. / Tetrahedron: Asymmetry 24 (2013) 324–333
lowed to stand at room temperature. When no reaction occurred
after 24 h at room temperature, toluene (40 ml) was added, and
the mixture was transferred to a Dean–Stark apparatus to be
heated at reflux. After 12 h, the reaction mixture was cooled to
room temperature, washed with saturated sodium bicarbonate
solution (20 ml), dried over MgSO4 and concentrated in vacuo.
Purification by column chromatography (RF = 0.67 in 1:5 EtAc/hex-
ane) yielded the title compound as a crystalline solid (0.45 g, 61%
chromatography (1:10 ethyl acetate in hexane, RF = 0.42) to give
the title compound (0.52 g, 59% over two steps) 24. Mp 57–60 °C.
[
a
]
D = À59.7 (c 1.0, dichloromethane). IR: v = 2927, 2854, 1737).
1H NMR (400 MHz) d 3.96 (d, 1H, J 2.1, CHO), 2.18 (m, 1H), 1.93
(m, 2H), 1.59 (m, 2H), 1.30 (s, 6H, CH3), 1.12 (s, 3H, CH3), 0.88 (s,
9H, SiC(CH3)3), 0.14 (s, 3H, CH3), 0.07 (s, 3H, CH3). 13C NMR
(100 MHz) d 208.5 (C@O), 76.8, 74.7, 73.7, 44.8, 29.6, 29.4, 27.2,
25.7, 22.6, 21.1, 19.3. À4.7, À5.3. m/z (%) 43 (29.8), 59 (11.3), 69
(14.3), 73 (56.6), 75 (100), 93 (11.8), 115 (11.9), 121 (35.7), 197
(57.5). [Found: C 64.5, H 10.2. C16H30O3Si requires C 64.4, H
yield). Mp = 78–79 °C. IR: v = 2975, 2906, 1740. [
a
]
D = À92.2 (c
1.0, ethanol). IR: v = 2976, 2901, 1742. 1H NMR (500 MHz) d 4.41
(m, 1H, CH2O), 4.10 (m, 2H, CH2O), 4.00 (m, 1H, CH2O), 2.17 (dddd,
1H, J 14.3, 11.5, 5.2 and 2.5, C7), 2.04 (app. tm, J 11.5 and 3.7, 1H
C8), 1.96 (ddd, 1H, J 14.3, 11.5 and 5.2, C7), 1.86 (m, 1H, methine),
1.74 (ddd, 1H, J 14.3, 11.6 and 3.6, C8), 1.38 (s, 3H, C10/11 CH3),
1.37 (s, 3H, C10/11 CH3), 1.11 (s, 3H, C9 CH3).). 13C NMR
(125 MHz) d 207.0 (C@O) 104.1 (acetal), 75.6 (C1), 74.2 (C3),
67.0, 63.7, 45.2 (C4), 30.0, 29.4, 28.7, 19.3 (C9), 17.9. NMR assign-
ments were made with the assistance of HSQC and HMBC. m/z
(%) 43 (73), 55 (38), 67 (11), 99 (16), 127 (100), 141 (21), 183
(10), 198 (10). [Found: C 63.6, H 8.2. C12H18O4 requires C 63.7, H
8.0.] [Found: m/z 198.1249. M+Å–CO requires m/z 198.1256.]
10.1.] [Found: m/z 321.1855 by ESI-HRMS.
requires m/z 321.1856.]
C16H30O3Si + Na
4.22. (1R)-5b-Hydroxy-6b-ketocineole 20
The TBDMS ether 24 (0.15 g, 0.50 mmol) was dissolved in
methanol (4 ml) and chilled on ice. Next, tert-butylammonium
fluoride (1 ml, 1 M in THF, 1 mmol) was added and the solution
was stirred at on ice for 15 min, after which water (10 ml) was
added. The aqueous mixture was extracted with ether
(2 Â 20 ml). The organic extracts were dried over magnesium sul-
fate, evaporated and purified by column chromatography (1:2
EtAc/benzene) to yield the title compound as a clear oil (0.038 g,
4.20. (1R)-6-(1,3-Dioxolan-2-yl)-5-ketocineole 22
41%). [
a
]
D = À5.5 (c 1.9, dichloromethane). 1H NMR (300 MHz,
(1R)-5,6-Diketocineole 11 (0.50 g, 2.7 mmol), p-toluenesulfonic
acid (10 mg), anhydrous copper(II) sulfate (10 mg) and ethylene
glycol (3 ml) were heated at reflux for 90 min. The reaction mix-
ture was cooled to room temperature, quenched with a saturated
sodium bicarbonate solution (15 ml) and extracted with hexane
(30 ml). The hexane extract was dried over MgSO4 and concen-
trated in vacuo, after which the crude extract was purified by col-
umn chromatography (RF = 0.51 in 1:5 EtAc/hexane) to yield the
title compound as a clear oil (0.13 g, 21%). Upon standing at 4 °C
fine needles of crystalline solid eventually formed, mp 43–44 °C.
C6D6) d 3.70 (d, 1H, J 2.0, CHO), 3.25 (br s, 1H, OH), 1.78 (m, 1H,
methine), 1.68 (m, 1H), 1.50 (m, 1H), 1.37 (d, 3H, J 0.5, CH3),
1.20 (s, 3H, CH3), 1.08 (br s, 3H, CH3), 1.02 (m, 1H), 0.99 (m, 1H).
13C NMR (75 MHz, C6D6) d 210.2 (C6), 76.7 (C5), 74.9 (C3), 73.2
(C1), 43.4 (C4), 29.7 (C10/11), 29.2 (C10/11), 26.9 (C7), 21.2 (C8),
19.2 (C9). m/z (%) 43 (100), 58 (41.8), 69 (25.8), 85 (17.8), 95
(18.2), 109 (12.1), 123 (15.2), 156 (6.4), 184 (M+Å, 3.6). Multiple
peaks were obtained by GC–MS; only the signal favoured by lower
injector port temperatures is reported.
IR: v = 2976, 1742. [
a
]D = +1.9 (c 1.0, ethanol). IR: v = 2975, 2906,
4.23. Crystallography
1740. 1H NMR (300 MHz) d 4.33 (m, 1H CH2O), 4.25 (m, 1H
CH2O), 4.04 (m, 2H CH2O), 2.20 (dd, J 6.7 and 2.4, 1H, methine),
2.14 (dm, J 6.7 and 17, 1H), 1.95 (m, 1H), 1.81 (m, 2H), 1.30 (s,
3H, CH3CCH3), 1.19 (s, 3H, CH3CCH3), 1.10 (s, 3H, C9 CH3). 13C
NMR (75 MHz) d 212.4 (C@O), 102.5 (acetal), 76.5 (C1), 73.9 (C3),
66.6, 66.4, 52.5 (C4), 30.0 (C10/11), 27.3 (C8), 27.2 (C10/11), 18.7
(C9), 18.1 (C7). NMR assignments were made with the assistance
of HSQC and HMBC. m/z (%) 43 (100), 55 (28), 69 (28), 73 (41),
83 (23) 112 (34), 127 (94), 141 (21), 183 (16), 226 (M+Å, 15).
[Found: C 63.9, H 8.0. C12H18O4 requires C 63.7, H 8.0.]
Data for 14, 16 and 21 were collected on an Enraf Nonius CAD4
diffractometer with Mo K
a radiation (0.71073 Å) operating in the
x
À 2h scan mode. Data reduction was performed with XCAD4.18
No absorption corrections were necessary. Due to crystal instabil-
ity at room temperature, data for compound 21 were collected at
223 K using an Oxford Cryosystems Series 600 Cryostream Cooler.
All other data were collected at 293 K. For compounds 17 and 22,
crystallographic data were collected on an Oxford Diffraction Gem-
ini Ultra
S
CCD diffractometer employing Mo
Ka radiation
(0.71073 Å) and operating in the
x
-scan mode. Data reduction
4.21. (1R)-5b-(t-Butyldimethylsilanoxy)-6-ketocineole 24
and empirical absorption correction (multiscans) were carried
out with the CrysAlisPro program (Oxford Diffraction, vers.
171.34.40).
(1R)-5b,6b-Dihydroxycineole 14 (0.55 g, 2.95 mmol) was trea-
ted with sodium hydride (0.25 g, 60% w/w oil suspension,
6.3 mmol) in diethyl ether (50 ml). Next, tert-butyl-dimethylsilyl
chloride (0.448 g, 2.97 mmol) was added. TLC monitoring indicated
that the reaction was complete after 5 min. Brine (10 ml) was
added and the ether layer was separated and dried over magne-
sium sulfate and the solvent removed in vacuo. The residue was
fractionated by flash chromatography (1:10 ethyl acetate in
hexane) to give a clear oil. This oil was dissolved in dichlorometh-
ane (40 ml) and added to a solution of oxalyl chloride (0.9 ml,
0.01 mol) and dimethyl sulfoxide (1.3 ml, 0.018 mol) at À78 °C.
The solution was stirred under a nitrogen blanket for 5 h, by which
time the temperature had risen to 0 °C. The solution was cooled to
À78 °C and quenched with triethylamine (6 ml, 0.04 mol). The
reaction was concentrated in vacuo, diluted with hexane (80 ml)
and washed with water (20 ml). The hexane extract was dried over
magnesium sulfate, concentrated in vacuo and purified by column
All structures were solved by direct methods using SHELXS86
and refined with SHELX97.19 All non-hydrogen atoms were refined
anisotropically except minor contributors to ketal ring disorder in
compound 22. All alkyl H-atoms were constrained at estimated
positions using a riding model while hydroxyl H-atoms were mod-
elled by allowing the H–O–C–C dihedral angle to refine to optimise
the position of the H-atom. All calculations were carried out within
the WinGX suite of programs20 and thermal ellipsoid diagrams
were generated with ORTEP3.21
Crystallographic data (excluding structure factors) for the
structures herein have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication nos.
CCDC 832941–832945. Copies of the data can be obtained, free of
charge, on application to CCDC, 12 Union Road, Cambridge CB2
1EZ, UK, (fax: +44 (0)1223 336033 or e-mail: deposit@ccdc.
cam.ac.uk).

A. J. Farlow et al. / Tetrahedron: Asymmetry 24 (2013) 324–333
333
4.23.1. Crystal data
References
Compound 14: C10H18O3: M 186.24, monoclinic, a = 6.193(1),
b = 12.329(1), c = 12.949(2) Å, b = 91.91(1)°, U = 988.2(2) Å3, space
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group P21 (No. 4), Z = 4, T = 293(2) K,
l(Mo Ka ,
) = 0.091 mmÀ1
Dc = 1.252 g cmÀ3, 1979 data (1804 unique, Rint = 0.037) were
measured in the range 3.14 < 2h < 50°, R = 0.0419 (for 1256 reflec-
tions with I >2
r(I)), wR2 = 0.1155 (all data). GoF = 1.061. CCDC No.
832941.
Compound
16: 10H18O3: 186.24, orthorhombic,
C
M
a = 9.8262(6), b = 9.970(1), c = 10.421(1) Å, U = 1020.9(2) Å3, space
group P212121 (No. 19), Z = 4, T = 293(2) K,
) = 0.088 mmÀ1 Dc = 1.212 g cmÀ3
1368 data (993 unique,
Rint = 0.0597) were measured in the range 5.66 < 2h < 50°,
l(Mo
Ka
,
,
R = 0.0364 (for 554 reflections with I >2r(I)), wR2 = 0.1075 (all
data). GoF = 0.976. CCDC No. 832942.
6. Slessor, K. E.; Hawkes, D. B.; Farlow, A.; Pearson, A. G.; Stok, J. E.; De Voss, J. J. J.
Mol. Cat. B: Enzym. 2012, 17, 15–20.
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Sons Ltd: West Sussex, 2007; Vol. 3, pp 397–435.
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Marburg, 1995.
Compound
a = 7.0664(6), b = 8.5413(6), c = 16.329(1) Å, U = 985.5(1) Å3, space
group P212121 (No. 19), Z = 4, T = 293(2) K, (Mo
) = 0.091 mmÀ1, Dc = 1.255 g cmÀ3, 2585 data (1034 unique,
Rint = 0.048) were measured in the range 6.28 < 2h < 50°,
17:
C
10H18O3:
M
186.24,
orthorhombic,
l
Ka
R = 0.0346 (for 522 reflections with I >2r(I)), wR2 = 0.0903 (all
data). GoF = 0.765. CCDC No. 832943.
Compound
a = 5.9626(6), b = 11.800(2), c = 15.990(2) Å, U = 1125.0(3) Å3,
space group P212121 (No. 19), Z = 4, T = 223(2) K, (Mo
) = 0.099 mmÀ1, Dc = 1.336 g cmÀ3, 1599 data (1144 unique,
Rint = 0.0762) were measured in the range 4.28 < 2h < 50°,
21:
C12H18O4:
M
226.26,
orthorhombic,
l
Ka
R = 0.0585 (for 763 reflections with I >2r(I)), wR2 = 0.1678 (all
data). GoF = 1.024. CCDC No. 832944.
Compound 22: C12H18O4: M 226.26, monoclinic, a = 10.896(1),
b = 7.7822(9), c = 14.441(2) Å, b = 99.74(1), U = 1206.9(2) Å3, space
group P21 (No. 4), Z = 4, T = 293(2) K,
l(Mo Ka ,
) = 0.093 mmÀ1
Dc = 1.245 g cmÀ3, 6175 data (2271 unique, Rint = 0.0513) were
measured in the range 5.72 < 2h < 50°, R = 0.0484 (for 1149 reflec-
19. Sheldrick, G. M. Acta Crystallogr. A 2007, A64, 112–122.
20. Farrugia, L. J. J. Appl. Cryst. 1999, 32, 837.
21. Farrugia, L. J. J. Appl. Cryst. 1997, 30, 565.
tions with I >2r(I)), wR2 = 0.1112 (all data). GoF = 0.778. CCDC No.
832945.
Acknowledgements
This work was supported by the Australian Research Council
(DP 110104455) and the Australian Postgraduate Research Award
to A.J.F.