Short Total Synthesis of Ajoene

Article abstract of DOI:10.1002/anie.201808605

We describe a short total synthesis of ajoene, a major biologically active constituent of garlic. The instability of allicin as the only other known alternative starting material has led to the development of a reliable procedure for the synthesis of ajoene from simple building blocks that is also suitable for upscale operations.

Full text of DOI:10.1002/anie.201808605

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Accepted Article
Title: Short Total Synthesis of Ajoene
Authors: Filipa Silva, Shaista S Khokhar, Danielle M Williams, Robert
Saunders, Gareth J S Evans, Michael Graz, and Thomas
Wirth
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201808605
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10.1002/anie.201808605
Angewandte Chemie International Edition
COMMUNICATION
Short Total Synthesis of Ajoene
Filipa Silva,[a] Shaista S. Khokhar,[b] Danielle M. Williams,[b] Robert Saunders,[b] Gareth J. S. Evans,[b]
Michael Graz,[b] and Thomas Wirth*[a]
Abstract: We describe a short total synthesis of ajoene, a major
biologically active constituent of garlic. The instability of allicin as the
only other known alternative starting material has led to the
development of a reliable protocol for the synthesis of ajoene from
simple building blocks also suitable for upscale operations.
propargylated to form thioether 4. The reaction of the hydroxy
group in 4 with 2-nitrophenyl selenocyanate and tributylphosphine
produced the selenide 6a (Scheme 2). Alternatively, dibromide 3
(R = Br) can be treated with the phenylselenide anion generated
in situ from diphenyl diselenide to afford bromide 5. Compound 5
was then used to synthesize the propargylic thioether 6b using
the same sequence of isothiouronium salt formation, hydrolysis
and propargylation. Overall yields for the reaction sequences to
6a and 6b are 29% and 63%, respectively. The selenium moiety
will serve as the handle to introduce an alkene through a
selenoxide elimination.
For a long time, garlic extracts and garlic-based products have
been used worldwide not only as food ingredients, but also as
medicine for the prevention of stroke, coronary thrombosis, and
atherosclerosis, as well as in the treatment of infections and
vascular disorders.[ 1 ] The therapeutic benefits of garlic are
manifold and relate to the high concentrations of organosulfur
compounds present in this plant. However, the instability of the
major component allicin 1 limits the commercial viability of garlic
extracts. Among other constituents of garlic, ajoene 2 derived
from allicin is biologically active and more stable.[2]
1. thiourea (80%)
2. KOH, propargyl
bromide (56%)
R
Br
HO
S
(R = OH)
3
4
SeCN
NO2
PBu3
(65%)
To our knowledge, there is only one reported synthesis of ajoene
2. Block et al. described the biomimetic thermal rearrangement of
allicin in aqueous acetone (Scheme 1),[3] recently this synthesis
has been extended to produce a trifluorinated analogue.[ 4 ]
Although the synthesis is a one-pot conversion, it suffers from low
yields (34%) due to the formation reactive sulfur-containing
intermediates leading also to side products. This reaction does
also not allow the synthesis of structurally modified or substituted
analogues. More recently, Hunter et al. reported a synthetic route
to prepare a range of ajoene derivatives, although this route could
not be used to synthesize ajoene 2 itself.[5]
NaBH4
PhSeSePh
(R = Br)
(92%)
1. thiourea (81%)
2. KOH, propargyl
bromide (84%)
Se
R'
S
PhSe
Br
5
6a (R' = NO2)
6b (R' = H)
Scheme 2. Synthesis of aryl propyl selenides 6.
O
S
O
S
acetone / water
64 ºC, 4 h
The next step of the synthesis involved the regioselective addition
of thioacetic acid to the terminal alkyne 6.[6] The reaction was
carried out by dissolving alkyne 6 in degassed toluene and
heating to 85 °C with a radical initiator added to the solution,
followed by the dropwise addition of thioacetic acid over 40
minutes using a syringe pump (Scheme 3). When ACCN [azobis-
(cyclohexanecarbonitrile)] was used as radical initiator,
compound 7a was obtained as 2:3 mixture of E/Z stereoisomers
in 50% yield (7b: 2:3 E/Z, 64%). For 7b the yield was slightly
improved to 71% when AIBN [azobis(isobutyronitrile)] was used
instead of ACCN. We could show that at this stage the separation
of the E and Z stereoisomers was possible by chromategraphy.
However, the mixture was used in the next reaction, as it is not
stereospecific.
S
S
S
1 allicin
2 ajoene
Scheme 1. Block’s synthesis of ajoene 2.
We present here an efficient total synthesis of ajoene 2. An
isothiouronium salt was prepared by reaction of bromide 3 (R =
OH) with thiourea, which was then hydrolyzed to the thiol and
[a]
F. Silva, Prof. Dr. T. Wirth
School of Chemistry
Cardiff University
Park Place, Main Building, Cardiff CF10 3AT (UK)
E-Mail: wirth@cf.ac.uk
1.2 eq. AcSH
radical initiator
SAc
[b]
Dr. S. S. Khokhar, D. M. Williams, Dr. R. Saunders, Dr. G. J. S.
Evans
Neem Biotech
Roseheyworth Business Park North
Abertillery NP13 1SX (UK)
Se
R'
S
Se
R'
S
toluene, 85 ºC
(50-71%)
6a (R' = NO2)
6b (R' = H)
7a (R' = NO2)
7b (R' = H)
Supporting information for this article is given via a link at the end of
the document.
Scheme 3. Radical addition of thioacetic acid to form derivatives 7.
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10.1002/anie.201808605
Angewandte Chemie International Edition
COMMUNICATION
The hydrolysis of 7 to the thioenolate was achieved with
potassium hydroxide in methanol and the subsequent
sulfenylation with thiosulfonic acid S-alkyl ester 8[7] occurred in
good yields to give compound 9. The reaction was performed at
–40 °C in order to avoid side reactions of the highly reactive
thioenolate. The reaction with compound 7a (2:3 mixture of E/Z
stereoisomers) afforded 9a in 73% yield with the same E/Z ratio.
When the reaction was performed with compound 7b (1:1 mixture
of E/Z stereoisomers), compound 9b was obtained in 87% yield
and the ratio of E/Z stereoisomers changed to 2:3. Since the
stereoisomers could be separated by chromatography, a reaction
with Z-7b was performed in order to verify if isomerization to the
E-isomer occurs even at very low temperatures. Indeed, the
reaction afforded 9b as a 3:2 mixture of E/Z stereoisomers in 85%
yield.
beside compound 10 in 33%, compound 11 in 17% (Table 1, entry
4). Interestingly, when the reaction of compound 7a was carried
out in the presence of NaIO4, compound 11 was isolated as a
major compound in 50% yield (Table 1, entry 5). meta-
Chloroperbenzoic acid (mCPBA) was also investigated as a
suitable oxidant and found to react giving comparable yields
(Table 1, entry 6).
Table 1. Optimization studies.
O
10
SAc
S
[O]
SAc
PhSe
S
+
7a
O
S
11
SAc
PhSe
Entry
Oxidation conditions
Yield [%]
10
7a
11
19
1. KOH, MeOH
STs
1
2
3
4
5
6
7
8
9
2 equiv. H2O2 (50% w/w), THF
0 ºC (1 h) – rt (2 h)
20
23
S
Se
R'
S
2.
S
7a (R' = NO2)
7b (R' = H)
8
3 equiv. H2O2 (50% w/w), THF
0 ºC (1 h) – rt (2 h)
21
-
20
12
35
9
25
9
CH2Cl2, –78 ºC
(73-87%)
9a (R' = NO2)
9b (R' = H)
4 equiv. H2O2 (50% w/w), THF
0 ºC (1 h) – rt (2 h)
O
S
2 eq. H2O2
S
2 equiv. UHP, CH2Cl2
0 °C (1 h) – rt (2 h)
6
-
6
S
THF, 0 ºC
(23-27%)
2 ajoene
2 equiv. NaIO4, CH3OH/H2O
0 °C (2 h), then rt (6 h)
2 equiv. m-CPBA, CHCl3
0 ºC (1 h) – rt (2 h)
50
16
-
Scheme 4. Synthesis of ajoene 2.
-
37
27
46
44
In the final step of the synthesis, compound 9 is treated with two
equivalents of 30% w/w hydrogen peroxide solution to form
ajoene 2 in a 2:3 mixture of E/Z stereoisomers in 27% (9a) and
23% (9b) yield. The selenide as well as the sulfide functionality is
2 equiv. H2O2 (50% w/w), CH2Cl2, 1.5 equiv.
DIPA, 0 ºC (1 h) – rt (2 h)
20
-
2 equiv. mCPBA, CH2Cl2, 2 equiv. DIPA
0 ºC (1 h) – rt (2 h)
-
oxidized and while the selenoxide is undergoing
a direct
2 equiv. mCPBA, CH2Cl2, 2 equiv. DIPA
0 ºC (1 h) – rt (24 h)
-
-
selenoxide elimination to form a double bond, the sulfoxide is
retained in the product molecule. The syn-elimination of alkyl aryl
selenoxides is an efficient synthetic procedure to form alkenes. It
is known that electron-withdrawing substituents on the aromatic
ring increase both the rate of elimination and the yield of the
alkene.[8] However, the use of the selenium derivative with an
electron-withdrawing substituent 9a did not show any advantage
when compared with compound 9b as the yields are almost
identical. A perselenenic acid byproduct might be able to catalyze
the oxidation to the sulfoxide.[9] The yields for the conversion from
9 to ajoene 2 were rather low at a 0.3 mmol scale.
Further optimization studies of the selenoxide elimination and
concomitant sulfur oxidation were carried out. For this study,
compound 7a was used as the model substrate to find suitable
reaction conditions. Product 10 can also be used as ajoene
precursor. Different oxidation conditions were investigated and
the results are presented in Table 1. The reaction of compound
7a with 2 equivalents of H2O2 (50% w/w) afforded products 10
(23%) and 11 (19%) (Table 1, entry 1). Increasing the amount of
oxidant to 3 or 4 equivalents did not improve the yield of
compound 10 and compound 11 was still isolated (Table 1, entries
2 and 3). The complex urea • hydrogen peroxide (UHP) was also
used as an alternative to aqueous hydrogen peroxide solution.
The reaction of compound 7a with 2 equivalents of UHP afforded
Independently of the reaction conditions, compounds 10 and 11
were the two major products formed and isolated. However,
smaller amounts of other non-identified side products were also
detected. Areneselenenic acid generated during the selenoxide
elimination is in equilibrium with its disproportionation products
(diaryl diselenide and areneseleninic acid). Under neutral or
acidic conditions they can react with alkenes leading to the
formation of side products. Side reactions can be suppressed by
addition of alkyl amines. When 1.5 equivalents of diisopropyl-
amine (DIPA) was added to the reaction with H2O2, the formation
of compound 11 was suppressed, but compound 10 was only
isolated in 27% yield. Adding 2 equivalents of DIPA to the reaction
with mCPBA not only stopped the formation of compound 11, but
also improved the yield of product 10 to 46% (Table 1, entry 8).
The same reaction conditions with a longer reaction time did not
affect the yield of compound 10 (Table 1, entry 9).
The complete synthesis of ajoene 2 was scaled up with slightly
different results being obtained.[10] The synthesis of 5 proceeded
with 58% yield on a 4 mol scale, while the subsequent thiol
formation and propargylation led to 6b in 87% (2.9 mol). Radical
addition of thioacetic acid proceeded similarly well compared to
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10.1002/anie.201808605
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COMMUNICATION
the small-scale synthesis (7b: 75%, 1.4 mol) as did the
thioacetate cleavage and thioallylation to 9b (74%, 1.1 mol). The
final oxidation to ajoene 2 had superior yields (65%) as compared
to the small scale synthesis and 169 g (0.72 mol) of ajoene 2 was
isolated in ~90% purity as determined by HPLC and NMR
analysis.
Two ajoene products were examined; 2 (synthetic) as
synthesized above and ajoene 2 (garlic) extracted from garlic
using the thermal rearrangement conditions.[3] the results are
expressed as a mixture of E and Z ajoene. Both ajoene samples
are effective QSIs as shown by their inhibition of the fluorescence
values in Figure 1, where a reduction in fluorescence is directly
related to the down regulation of the QS gene lasB.
Much of the research interest in ajoene 2 resides in its biological
activity. It has been shown to have efficacy in a number of
biological studies that include antithrombotic and antifungal
activities.[11] In order to further evaluate 2, its activity in a biological
assay was also examined. Ajoene’s ability to act as a quorum
sensing inhibitor (QSI) was selected, as this is one of its more
recent remarkable biological properties. Quorum sensing (QS) is
a mechanism of cell-cell communication in bacteria facilitated by
the secretion and detection of signalling molecules such as N-acyl
homoserine lactones in Gram-negative bacteria.[12] QS allows
bacteria to synchronise specific gene expression which has an
impact on their pathogenicity and is thought to have a significant
role in the formation of biofilms. Recent studies have shown that
ajoene 2 is an effective QS inhibitor against Pseudomonas
aeruginosa and Staphylococcus aureus and could be utilised for
the treatment of chronic biofilm infections by exploiting the QS
system.[13] In this study, we employed a reporter strain (PaO1-
lasB-gfp)[13a] whereby QS gene expression was monitored over
time in response to ajoene treatment.
The samples show a very similar pattern of concentration-
dependent inhibition. This is reiterated in the IC50 calculations
where ajoene 2 (garlic) extracted from garlic had an IC50 value of
27.7 µM and synthetic ajoene 2 (synthetic) had an IC50 value of
28.5 µM. The IC50 values are comparable between the different
origins of ajoene 2.
In conclusion, we describe an efficient total synthesis of ajoene
from easily available starting materials. The simultaneous
introduction of the allyl moiety and the sulfoxide in the final step
allows a straightforward generation of the target molecule. Up-
scaling of the synthetic sequence was possible leading for the first
time to the synthesis of synthetic ajoene in larger amounts.
Synthetic ajoene and ajoene derived from garlic have been
investigated regarding their efficiency as quorum sensing
inhibitors.
Experimental Section
The vinyl disulfide 9a (0.140 g, 0.33 mmol) is dissolved in THF (3 mL) and
cooled to 0 °C under N2, and H2O2 (30% w/w in H2O, 0.075 mL, 0.66 mmol)
added dropwise. The mixture was allowed to stir for 1 h at 0 °C and then
warmed to rt (2 h). Sat. aq. NaHCO3 (5 mL) was added and the residue
was extracted with EtOAc (2 × 10 mL). The combined organic fractions
were washed with brine (2 × 10 mL) and dried over MgSO4. The solvent
was removed under vacuum and the resulting residue purified by column
chromatography to afford ajoene 2 (21 mg, 27%, E/Z = 1:1.8) as a pale-
yellow oil.
100%
80%
60%
40%
20%
0%
Acknowledgements
We thank Neem Biotech Ltd. and EPSRC for support and the
EPSRC National Mass Spectrometry Facility, Swansea, for mass
spectrometric data.
2 (synthetic)
75 µM 37.5 µM
2 (garlic)
18.75 µM
Keywords: ajoene • allicin • garlic • organosulfur compounds •
Figure 1. Inhibition of a PaO1 lasB-gfp reporter strain, where inhibition of
selenoxide elimination
fluorescence is directly related to QS-controlled expression,
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10.1002/anie.201808605
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COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Making garlic: An efficient total
synthesis of ajoene from easily
available starting materials is
reported.
F. Silva, S. S. Khokhar, D. M. Williams,
R. Saunders, G. J. S. Evans, M. Graz, T.
Wirth*
AcSH
STs
PhSe
H2N
Br
S
Page No. – Page No.
Br
NH2
Short Total Synthesis of Ajoene
O
S
S
S
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