New excursions into the synthesis and medicinal chemistry of the disulfide bond

Article abstract of DOI:10.1080/10426507.2012.736107

The presentation discusses two themes of current interest in my research group in conjunction with a garlic mimics program. The first involves a new one-pot methodology for preparing unsymmetrical disulfides in high yield using 1-chlorobenzotriazole (BtCl) as oxidant, which can be applied to a range of functional types. The second part of the lecture will discuss the synthesis of derivatives of the garlic-derived natural product ajoene, in conjunction with an anti-cancer screening program. The synthesis has been used to prepare a fluorescently tagged derivative of the natural product that has been used to investigate localization and mode of action within the cancer cell using confocal laser scanning microscopy.

Full text of DOI:10.1080/10426507.2012.736107

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Phosphorus, Sulfur, and Silicon and the
Related Elements
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New Excursions Into the Synthesis and
Medicinal Chemistry of the Disulfide
Bond
Roger Hunter a , Catherine Kaschula a , Nashia Stellenboom a ,
Jonathan Cotton a & M. Iqbal Parker a
a Department of Chemistry , University of Cape Town , Rondebosch ,
South Africa
Accepted author version posted online: 24 Oct 2012.Published
online: 20 Sep 2013.
To cite this article: Roger Hunter , Catherine Kaschula , Nashia Stellenboom , Jonathan Cotton
& M. Iqbal Parker (2013) New Excursions Into the Synthesis and Medicinal Chemistry of the
Disulfide Bond, Phosphorus, Sulfur, and Silicon and the Related Elements, 188:11, 1497-1507, DOI:
10.1080/10426507.2012.736107
To link to this article: http://dx.doi.org/10.1080/10426507.2012.736107
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Phosphorus, Sulfur, and Silicon, 188:1497–1507, 2013
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2012.736107
NEW EXCURSIONS INTO THE SYNTHESIS AND
MEDICINAL CHEMISTRY OF THE DISULFIDE BOND
Roger Hunter∗, Catherine Kaschula, Nashia Stellenboom,
Jonathan Cotton, and M. Iqbal Parker
Department of Chemistry, University of Cape Town, Rondebosch, South Africa
GRAPHICAL ABSTRACT
Abstract The presentation discusses two themes of current interest in my research group in
conjunction with a garlic mimics program. The first involves a new one-pot methodology for
preparing unsymmetrical disulfides in high yield using 1-chlorobenzotriazole (BtCl) as oxidant,
which can be applied to a range of functional types. The second part of the lecture will discuss
the synthesis of derivatives of the garlic-derived natural product ajoene, in conjunction with an
anti-cancer screening program. The synthesis has been used to prepare a fluorescently tagged
derivative of the natural product that has been used to investigate localization and mode of
action within the cancer cell using confocal laser scanning microscopy.
Keywords Disulfide; heterodisulfide synthesis methodology; ajoene; cancer; fluorescently
tagged; CLSM; ER and mitochondrial stress
INTRODUCTION
The disulfide functionality1 plays a pivotal role in biochemical systems and is found
in some natural products. Some years ago, we initiated a program aimed at exploring the
medicinal chemistry of garlic mimics and an example of a relatively stable thiosulfinate
included into the cyclodextrin TRIMEB as an allicin mimic is shown in Figure 1.2 This
lecture describes various aspects of the chemistry that has emerged from this program.
In the first part, an overview is given on a new methodology for unsymmetrical disulfide
synthesis, while in the second part attention is directed toward chemical biology aspects of
the garlic-derived natural product ajoene.
Received 25 September 2012; accepted 25 September 2012.
This work was supported by the South African Research Chairs Initiative (SARCHI) of the Department
of Science and Technology, the National Research Foundation (NRF), and research grants from the Medical
Research Council (MRC) of South Africa and the University of Cape Town.
Address correspondence to Roger Hunter, Department of Chemistry, University of Cape Town, Rondebosch,
7701, South Africa. E-mail: roger.hunter@uct.ac.za
1497

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R. HUNTER ET AL.
Figure 1 S-p-anisyl 1-propylthiosulfinate2a included into TRIMEB. (Color figure available online).
RESULTS AND DISCUSSION
A New Heterodisulfide Synthesis
The efficient oxidation of two different thiols to a heterodisulfide (unsymmetrical
disulfide) is a subtle methodological challenge that has been addressed by several different
methodologies over the years.3 By far the most studied strategy3a is that involving thiolysis
of a sulfenyl derivative, in which the challenge is to engineer a sufficient rate difference
between reaction of R1SH to form the sulfenyl derivative R1SLG over that of reaction of
R1SLG with R1SH to form homodimer 1 as a byproduct (i.e., k1/k2 needs to be high).
Another irritating side reaction can be the exchange of the heterodimer with R2SH to form
homodimer 2, a reaction that is well documented for aromatic disulfides, Scheme 1.
Scheme 1 Side reactions in the sulfenyl methodology for heterodisulfide synthesis.
Over the years, many reagents have been introduced to produce a range of sulfenyl
derivatives bearing different leaving groups, but a novel way of addressing the rate difference
issue has not been addressed. Furthermore, reagents for producing the sulfenyl derivative
are not always benign. Scheme 2 shows four such popular methodologies for sulfenyl
intermediate production based on sulfur-based leaving groups, all of which have varying
drawbacks along the lines just discussed.

SYNTHESIS AND MEDICINAL CHEMISTRY
1499
Scheme 2 Various popular sulfenyl derivatives involving sulfur-based leaving groups.
Considering this problem a few years ago we were inspired by the work of Abe,4
who showed that N-chlorosuccinimide reacts with a thiol to form the sulfenyl chloride
accompanied unsurprisingly by significant amounts of homodimer 1. However, on addition
of triethylamine the sulfenyl chloride was transformed via base-catalyzed substitution into
the more stable sulfenimide, Scheme 3.
Scheme 3 Abe’s sulfenimide synthesis.4
We rationalized that a more nucleophilic leaving group might improve the afore-
mentioned k1/k2 ratio by converting the sulfenyl chloride, as it formed rapidly into a less
reactive “masked” sulfenyl derivative. The chlorinating agent eventually chosen for the
purpose was 1-chlorobenzotriazole, a reagent introduced by Rees5 in the late 1960s as an
oxidant of primary alcohols, which is readily prepared by oxidation of benzotriazole with
bleach. It was thought that the superior nitrogen nucleophilicity of benzotriazole over that
of succinimide would promote trapping and in a model reaction involving p-anisylthiol,
we were gratified to achieve a high yield of the heterodisulfide (89%) with no formation
of ho◦modimer 1. Formation of the intermediate R1SBt derivative proceeded efficiently at
◦
−78 C, as evidenced by TLC, while reaction with R2SH was conducted at −20 C. An
equivalent of BtH was added in the reaction to assist with trapping, and BtH could be
recovered in the work-up using an acid–base extraction. A small percentage of homodimer
2 was produced based on the reagent loadings, Scheme 4.
The observed chemoselectivity was rationalized on the basis of in situ trapping of the
sulfenyl chloride to a much less reactive Bt sulfenamide, Scheme 5.
A range of thiols has been subjected to Bt-methodology verifying the broad scope
of the method. Thus, the method works well for aromatic disulfide synthesis without
an exchange processes producing homodimer 2. Aromatic-alkyl combinations are also

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R. HUNTER ET AL.
Scheme 4 BtCl methodology for heterodisulfide synthesis.
Scheme 5 Mechanistic rationale of chemoselectivity.
produced efficiently as in Scheme 4 in which it is customary to add the aliphatic thiol
as R2SH so as to be able to chromatograph homodimer 2 in the non-polar fraction. In
all cases, the production of homodimer 1 is minimal to nothing. For aliphatic disulfides,
excess reagent can be neutralized by thiourea before adding R2SH, resulting in a final
heterodisulfide product devoid of either homodimer byproduct. These reactions all utilized
the −78 ◦C/−20 ◦C combination of the prototype. Similarly, the methodology was found
to be extendable to cysteine as well as sugar-based couplings. In this instance, the preferred
reaction temperature was found to be −78 ◦C throughout. Figure 2 shows a broad range of
disulfides synthesized by our in situ trapping methodology.3b,6
In summary, a new methodology for heterodisulfide synthesis has been developed
involving a novel in situ trapping aspect that minimizes formation of homodimer 1 as a
byproduct. The method covers a broad range of target, and is relatively green. The first step
is not compatible with protic solvents in view of the intermediacy of a sulfenyl chloride,
while the thiolysis step converting R1SBt to product can withstand a level of polarity in the
medium, eg is compatible with methanol. It is hoped that the method can be adapted overall
for polar biomolecule systems, probably by using a dipolar aprotic solvent like DMF in the
first step.
The Chemical Biology of Ajoene
Ajoene is a natural product found as a rearrangement product in freshly-crushed garlic
and was first reported in the literature in the 1980s by Apitz-Castro and Block,7 who in
their seminal paper proposed a biosynthesis involving Michael addition to a vinylthionium
ion. The salient details are summarized in Scheme 6.
Since its discovery, ajoene has been shown to possess a range of biological activities
including antimicrobial,8 antiobesity,9,10 antifungal,11–14 and anticancer,15–17 with the latter
of particular interest to my group. Ajoene has been shown to inhibit tumor cell prolif-
eration by inducing apoptosis in a number of cell lines, with IC50’s ranging from 5 μM
to 40 μM.18–20 In vivo, ajoene has been shown to inhibit the growth of skin carcinoma
in both rodents and humans,21,22 and to inhibit tumor cell growth in mouse xenografts by

SYNTHESIS AND MEDICINAL CHEMISTRY
1501
Figure 2 Scope of the BtCl methodology.
2–3-fold.18,19 A biomimetic synthesis of ajoene has been described by Block,7 but this is not
amenable to derivative production. We have recently23 developed an ajoene synthesis that
allows modification of the end-groups while retaining the vinyl disulfide pharmacophore,
Scheme 7. In such a way, we have been able to improve antiproliferation activity (IC50) in
an oesophageal cell-line relative to Z-ajoene 12-fold by substituting the allyl end groups
Scheme 6 Biosynthesis of allicin and ajoene.

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R. HUNTER ET AL.
Figure 3 Structure-activity hypothesis for Z-ajoene in its inhibition of WHCO1 cancer-cell proliferation.
with p-methoxybenzyl groups. Overall, structure-activity studies have revealed the profile
shown in Figure 3.24
Several groups have uncovered aspects of mode of action and molecular targets
of ajoene, but a comprehensive picture has yet to emerge. Ajoene and the related garlic
organosulfur compounds diallyl disulfide and diallyl trisulfide have been shown to induce
cell-cycle arrest in the G2/M phase of the cell cycle.25,26 Apoptosis has also been demon-
strated to proceed via the mitochondrial-dependent caspase cascade with involvement of
Scheme 7 Synthesis of end-substituted ajoene analogues.

SYNTHESIS AND MEDICINAL CHEMISTRY
1503
mitochondrial membrane permeabilization (MMP), release of cytochrome c, caspase 3 ac-
tivation, and cleavage of the anti-apoptotic protein Bcl-2.19,26–32 It has been proposed33–35
that the antiproliferation activity of garlic organosulfur compounds may be tied to the
chemical reactivity of the disulfide bond, which may thiolate cysteine residues in proteins.
In support of this hypothesis, the related garlic compound diallyl trisulfide has been found
to oxidatively modify β-tubulin in a cell-free environment to form mixed disulfides at
cysteine residues Cys-12β and Cys-354β.36 In another cell-free model, ajoene was found
to act as a covalent inhibitor and substrate of glutathione reductase through mixed disulfide
formation with Cys58 in the active site of the enzyme.37 Thus, a key question remains:
what gives rise to the primary mitochondrial stress and where does it take place within the
cell? In view of our access to end-substituted ajoenes through total synthesis, we decided
to address this chemical biology question by fluorescently tagging the ajoene skeleton in
which it was decided to incorporate a dansyl tag in view of its ready availability and its λem
being in the visible range at around 500 nm in accordance with requirements of our CLSM.
Since our previous study had demonstrated that the allyl end groups of ajoene could be
substituted without loss of bioactivity, we decided to substitute one of the allyl groups by
a propyldansyl tag, which was chosen for the vinyl disulfide side. On the sulfoxide side, a
propyl was identified in view of its similarity to allyl; allyl could not be accommodated in
our synthesis because of complications occurring in the radical addition step. In the event,
the synthesis of our dansyl-tagged ajoene turned out to be relatively straightforward and
is shown in Scheme 8. Thus, 1-propylthiol was propargylated under standard alkylation
conditions to the sulfide, which underwent a regioselective and moderately stereoselec-
tive radical addition with thiolacetic acid to furnish vinyl acetate 2 as a 3:5 E:Z-mixture
of stereoisomers. Thereafter, the dansyl-containing tether 3 was routinely prepared from
3-aminopropanol via a dansylation, mesylation, iodination, and substitution sequence in-
volving thiotosylate as nucleophile in the final step. Low temperature base cleavage of
vinylthioacetate 2 to its enethiolate, followed by S-thioalkylation with 3, furnished the
disulfide, which was chemoselectively mono-oxidized at the sulfide sulfur to afford the
target ajoene derivative 4 as a 3:5 E/Z-mixture of stereoisomers that could not be sepa-
rated by chromatography. Yields of all steps were above 65% and all intermediates gave
an appropriate and satisfactory set of analytical data (see experimental section) in which
individual geometrical isomers could be easily distinguished by the coupling constant of
the vinyl hydrogen attached to the α-carbon to the vinyl disulfide. We were not fazed by
not having separate geometrical isomers, since previous studies8,19,23 had shown that both
were biologically active against cancer cells, albeit with Z- generally a little more active
Scheme 8 For 4: (a) KOH, MeOH, 20◦C, propargyl bromide; (b) AcSH, toluene, ACCN (10 mol%), 80◦C, 2 h;
(c) KOH, MeOH, −40◦C, 30 min followed by 3; (d) m-CPBA, CH2Cl2, −60◦C, 2 h. For sulfenylating agent 3:
(i) Dansyl Cl, CH2Cl2, 0◦C, 2 h; (ii) MsCl, NEt3, CH2Cl2, 0◦C, 2 h; (iii) NaI, CH3CN, 60◦C, 18 h; (iv) KSTs,
CH3CN, 20◦C, 18 h.

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R. HUNTER ET AL.
Figure 4 Live cell imaging (CLSM) of live MDA-MB-231 cells treated with DP (blue) for 345 min. ER stained
with ER-tracker (RED). (Color figure available online).
than E-. Certainly in the context of the present study, this aspect was not deemed to be
limiting; thus, the E/Z-mixture was used as isolated.
The antiproliferation activity of tagged derivative 4 was measured against the MDA-
MB-231 cell-line, a line of neoplastic breast-cells that were selected for the microscopy
because they are spread out and have a large cytoplasm ideal for viewing under the mi-
croscope. Against this cell-line the tagged derivative proved to be marginally less potent
than the natural product ajoene itself, returning an IC50 of about 21 μM versus the natural
Figure 5 Two possible mechanisms of action of ajoene within the ER.

SYNTHESIS AND MEDICINAL CHEMISTRY
1505
product itself (Z-ajoene = 13.7 2.0; E-ajoene = 18.6 5.5). Thus, the stage was set for
the confocal laser scanning microscopy (CSLM) experiment. Several fluorescent organelle
stains were used, namely: ER-tracker Red (Invitrogen E34250), Lyso-tracker Red (Invitro-
gen L7528), Mito-tracker Green (Invitrogen M7514), and DAPI (Invitrogen D3571). ER-,
Lyso-, and mito-tracker are live cell dyes, which accumulate in living cells and stain specific
organelles. The nuclear stain DAPI does not permeate living cells, but stains the nuclei of
dead cells and was hence used to differentiate living from dead cells. The cells were grown
on a chambered cover glass and kept in an incubated viewing chamber (CO2 = 5%, T =
37.5◦C) on the microscope. The fluorescent ajoenes were administered at 40 μM and the
various organelle trackers were used as recommended by the manufacturer (see experi-
mental section) to observe live cell imaging. The dansyl fluorophore is typically excited at
around 350 nm, which lies within the UV region of the electromagnetic spectrum, which is
not suitable for studying live cells. UV-light would render any observations of localization
invalid as the cells would be damaged by this radiation. Using a 2-photon excitation how-
ever, it was possible to excite this fluorophore with near infrared light (700 nm), which is
not harmful radiation to the cells. Various organelles were studied regarding the possibility
of colocalization with tagged derivative 4 (seen as a coalescence of the colors of both
fluorescent ajoene [blue] and tracker), and eventually it became clear that coalescence was
only observed within the endoplasmic reticulum (ER) as shown in Figure 4. This result
was confirmed using a yellow fluorescent protein cloned into the ER, where once again
coalescence was noted with dansyl derivative 4.
CONCLUSIONS
This lecture has presented results on a novel methodology for heterodisulfide syn-
thesis that owes its novelty to trapping of the intermediate sulfenyl chloride. In such a
way, it achieves total suppression of homodimer 1 formation, as well as offers relatively
green options for accessing a broad product range. In the second part of the lecture, a novel
four-step synthesis has been presented that allows access to end-substituted derivatives of
the rearrangement product of freshly crushed garlic ajoene. A library of such derivatives
has been evaluated against various cancer cell-lines for their antiproliferation activity, and
a structure-activity profile has been generated. A key chemical biology question pertaining
to ajoene’s site of action within the cancer cell resulting in intrinsic mitochondrial stress
ultimately leading to apoptosis, has been addressed using a fluorescently tagged dansylated
ajoene in conjunction with confocal laser scanning microscopy. The results point toward
the ER being the likely site of ajoene’s biochemical activity within the cancer-cell. Given
the involvement of the ER in cancer-cell disulfide synthesis, a potential interpretation of
mechanism of action33,37 resulting in mitochondrial stress leading ultimately to apoptosis is
given in Figure 5. Future work will continue probing the target of ajoene bioaction within
the ER.
EXPERIMENTAL
A full experimental set of data will be published elsewhere. For 4: (E/Z) mixture:
Rf = 0.2 (EtOAc:Hexane = 1:1); νmax/cm−1(CHCl3): 1142 (sulfonamide), 1010 (sulfoxide),
541 (S-S); HRMS (EI): m/z 487.1221 [M +H]+, C21H31N2O3S4 requires 487.1218. E-
isomer: δH (400 MHz, CDCl3): 8.54 (1H, d, J = 8.5, Hz, ArH), 8.34 (1H, m, ArH), 8.22
(1H, m, ArH), 7.54 (2H, m, ArH), 7.19 (1H, m, ArH), 6.34 (1H, d, J = 14.7 Hz, H-4), 6.06

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R. HUNTER ET AL.
(1H, t, NH), 5.93 (1H, dt, J = 14.7, 7.4 Hz, H-5), 3.53 (1H, dd, J = 13.2, 8.4 Hz, H-6a),
3.38 (1H, dd, J = 13.2, 8.4 Hz, H-6b), 2.99 (2H, q, J = 6.5 Hz, H-1), 2.89 (6H, s, NMe2),
2.79–2.59 (4H, m, H-3 and H-7), 1.81 (4H, m, H-2 and H-8), 1.08 (3H, t, J = 7.4 Hz, H-9);
δC (100 MHz, CDCl3): 151.9 (ArCqu), 135.2 (ArCqu), 134.6 (C-4), 130.2 (ArC), 130.0
(ArCqu), 129.7 (ArCqu), 129.3 (ArC), 128.2 (ArC), 123.2 (ArC), 119.1 (ArC), 117.5 (C-5),
115.2 (ArC), 53.9 (C-6), 53.3 (C-7), 45.4 (NMe2), 41.5 (C-1), 35.7 (C-3), 29.0 (C-2), 16.4
(C-8), 13.4 (C-9). Z-isomer: δH (400 MHz, CDCl3): 8.54 (1H, d, J = 8.5, Hz, ArH), 8.34
(1H, m, ArH), 8.22 (1H, m, ArH), 7.54 (2H, m, ArH), 7.19 (1H, m, ArH), 6.46 (1H, d, J =
9.4 Hz, H-4), 5.73 (1H, dt, J = 9.4, 8.2 Hz, H-5), 5.63 (1H, t, NH), 3.57 (2H, d, J = 8.9 Hz,
H-6a and H-6b), 2.99 (2H, q, J = 6.5 Hz, H-1), 2.89 (6H, s, NMe2), 2.79–2.59 (4H, m, H-3
and H-7), 1.81 (4H, m, H-2 and H-8), 1.08 (3H, t, J = 7.4 Hz, H-9); δC (100 MHz, CDCl3):
152.0 (ArCqu), 137.8 (C-4), 134.9 (ArCqu), 130.4 (ArC), 130.0 (ArCqu), 129.7 (ArCqu),
129.5 (ArC), 128.3 (ArC), 123.2 (ArC), 119.5 (C-5), 118.9 (ArC), 115.2 (ArC), 53.6 (C-7),
50.9 (C-6), 45.4 (NMe2), 41.6 (C-1), 35.6 (C-3), 28.9 (C-2), 16.4 (C-8), 13.4 (C-9).
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