Synthesis of 1, 1′, 2, 2′, 3, 3′, 4, 4′ - Octadeutero-Sulforaphane

Article abstract of DOI:10.1002/jlcr.833

Sulforaphane (SFN), a naturally occurring isothiocyanate present in broccoli, shows strong evidence of anti-carcinogenic activity. The mechanism of action, absorption, distribution, metabolism and excretion of the compound is however still poorly understood and requires a stable isotope labelled version of the compound for further studies. The paper describes an optimized synthesis of octadeutero-SFN.

Full text of DOI:10.1002/jlcr.833

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 501–507.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.833
Research Article
Synthesis of 1, 10, 2, 20, 3, 30, 4, 40 - Octadeutero-
Sulforaphane
Nikolai Kuhnert* and Ye Lu
Department of Chemistry, School of Biomedical and Molecular Sciences, University of
Surrey, Guildford GU2 7XH, UK
Summary
Sulforaphane (SFN), a naturally occurring isothiocyanate present in broccoli, shows
strong evidence of anti-carcinogenic activity. The mechanism of action, absorption,
distribution, metabolism and excretion of the compound is however still poorly
understood and requires a stable isotope labelled version of the compound for further
studies. The paper describes an optimized synthesis of octadeutero-SFN.
Copyright # 2004 John Wiley & Sons, Ltd.
Key Words: deuterium; isothiocyanate; anti-cancer compound
Introduction
The inverse relationship between vegetable and fruit consumption and the
incidence of various cancers in humans is well established.1 It is not only the
high concentration of vitamins, minerals and fibres that are responsible for
these effects but plant derived compounds refered to as phytochemicals.2
One major subgroup of phytochemicals are the glucosinolates (GLS) e.g.
Glucoraphanin 1. GLS per se are biologically inactive but following cell
disruption, the GLSs undergo enzymatic or non-enzymatic hydrolysis3–5 to
give as major products isothiocyanates (e.g. sulforaphane) to which the
anticarcinogenic effects of broccoli-rich diets have been attributed.6,7 As a
result of a statement by the National Cancer Institute SFN is one of the 40
most promising anticarcinogens.1 Based on epidemiological data1,8 and
experimental findings from numerous in vivo and in vitro studies on the
biological activity of SFN,9–11 there is a great interest in this particular
isothiocyanate, which is found mainly in broccoli.12,13
*Correspondence to: N. Kuhnert, Department of Chemistry, School of Biomedical and Molecular Sciences,
University of Surrey, Guildford GU2 7XH, UK. E-mail: n.kuhnert@surrey.ac.uk
Contract/grant sponsor: Association for International Cancer Research
Copyright # 2004 John Wiley & Sons, Ltd.
Received 18 September 2003
Revised 3 April 2004
Accepted 15 April 2004

502
N. KUHNERT AND Y. LU
OH
O
O
O
S
HO
HO
Myrosinase
or H+
S
S
SCN
OH
-O3SO
N
Sulforaphane 2
Glucoraphanin 1
Using different models it has been shown that SFN is a very potent phase II
enzyme inducer particularly of the glutathione S-transferase the principal
enzyme system involved in the detoxification of carcinogenic chemicals. It has
also been shown to suppress the activity of cytochrome P-450 enzymes
involved in the bioactivation of chemical carcinogens. In this way it alters the
balance of phase I/phase II enzyme metabolism in such a way that the
activation of carcinogens during phase I is decreased and the rate of
detoxification is increased. That is why SFN may play an important role in
cancer prevention.14–16 Recent results show that SFN is also able to induce cell
cycle arrest and apoptosis in HT29 human colon cancer cells17 and induce an
anti-inflammatory response.18 Thus SFN belongs to a small group of
compounds enabling dual inhibitory action in carcinogenesis. However, the
mechanisms of absorption, bioavailibility and metabolism are poorly under-
stood. In order to address these questions related to bioavailability and
mechanism of biological effects there is a need for stable isotope labelled SFN,
which will enable us to
(1) Identify possible metabolites and conjugates with biological (macro)
molecules e.g. enzymes, peptides, amino acids.
(2) Obtain pharmacokinetic data on absorption and distribution.
Results and discussion
Four chemical syntheses of sulforaphane 2 have been reported to date.19–22
Our synthetic strategy for the synthesis of a stable isotope labelled 2 follows
our published procedure concerned with the synthesis of radiolabelled SFN.21
We reasoned that for the required mass spectrometric analysis of SFN and
metabolites a high number of deuterium labels would be advantageous.
Furthermore, the labels should be placed in positions that are not prone to H/
D exchange. Hence, we decided to place the deuterium labels in the four
carbon chain separating the NCS and methylsulfinyl moieties. We started our
synthesis with d8-tetrahydrofuran as a commercially available precursor.
Using standard conditions we transformed d8-THF 3 into d8-1,4 dibromobu-
tane 4. Standard Gabriel procedure afforded the d8-N-(4-bromo-butyl)
phthalimide 5 in good yield without considerable H/D exchange, despite the
strongly basic conditions employed.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 501–507

SYNTHESIS OF 1, 10, 2, 20, 3, 30, 4, 40 -
503
O
NH
O
D
D
D
D D
D D
O
D D
D D
48 % HBr
D
D
O
Br
Br
N
D
D
Br
D D
D
K2CO3
200 oC
D D
D
D
D
D
O
4
5
99 % isotopic purity
3
Nucleophilic substitution of bromide 5 with NaSMe afforded the d8-
thioether 6 in good yield and excellent isotopic purity. Oxidation of 6 using
sodium periodate in MeOH was shown to be the condition of choice to yield
the sulfoxide 7 in almost quantitative yield. Cleavage of the phthalimide using
hydrazine was uneventful to give the amine 8, which was isolated as its
hydrochloride.
For the final transformation to give sulforaphane thiophosgene and 3
equivalents of NaOH in a diethylether/water two phase solvent system gave
the title compound in good overall chemical and labelled yield.
O
O
O
S
D
D
D
D
D
D
D
D
D
D
D
(ii)
(i)
SCH3
N
CH3
N
5
D
D
D D
D
O
O
7
6
(v)
(iii)
D
(iv)
[Cl-]+H3N
D
D
D
D
O
S
O
S
D
D
D
D
D
D
D
D
D D
S
D
N
CH3
N
CH3
CH3
C
D
D
C
S
D
D
D D
D
D
S
[D-8]- 9
Erucin
[D-8]- SFN 2
8
Reagents and conditions: (i) NaSMe/EtOH; (ii) NaIO4/MeOH; (iii) 1.
H4N2/EtOH, 2. HCl; (iv) Cl2C ¼ S, NaOH; (v) 1. H4N2/EtOH, 2. HCl, 3.
Cl2C ¼ S, NaOH.
Yields and analytical data are summarized in Table 1. With the exception of
5 all unlabelled compounds have been described in the literature.19,20 Both 423
and 520 have been prepared according to literature procedures. We report the
analytical data of the labelled materials. All stable isotope labelled compounds
displayed a characteristic C–D stretching frequency in the IR spectra and
quintets in the 13C-NMR spectra for the corresponding CD2 carbons in the
positions expected.
The deuterium incorporation and extent of labelling was confirmed with a
variety of analytical methods. Firstly mass spectra did give the total deuterium
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 501–507

504
N. KUHNERT AND Y. LU
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 501–507

SYNTHESIS OF 1, 10, 2, 20, 3, 30, 4, 40 -
505
D
D D
R2N
D
D
D
D
D
1
1
X
X
+
4
R2N
D
4
D D
D
D D
D
D
A
B
4-9
Scheme 1. Fragmentation of deuterated compounds 4–9
Table 2. Mass spectral data and isotopic purity
Compound
Mass
(m/z)a
Total %
deuterationb
Deuteration
at C-1 (%)c
Deuteration
at C-4 (%)c
4
5
6
7
8
2
9
C4D8Br2
222, 224, 226
265, 267
233
>99
>99
97
95
95
99
99
96
94
94
90
93
99
99
97
95
96
93
95
C10H4D8BrNO2
C11H7D8NO2S
C11H7D8NO3S
C5H5D8NOS
C6H3D8NOS2
C6H3D8NOS2
249d
144
185
169
92
94
a All mass spectra were obtained using CI and isobutane as the ionizing gas.
b d8 - is defined as 100% and d0 as 0% deuteration.
c As estimated from the fragment ions A and B.
d Molecular ion [M+H]+
.
content of each individual compound. A direct comparison with a simulated
mass spectrum and use of the established software (Thermo-Finnigan
standard software was used.) did allow estimation of the total deuterium
incorporation. Analysis of the fragment ions as well as integration of signals in
the 1H NMR spectra at 500 MHz enabled the determination of the deuterium
incorporation at the C1 and C4 sites. The typical fragment ions allowing this
estimation arise from a cleavage a to the nitrogen functionality resulting in an
R2NCD2 fragment A and a second molecular ion B (Scheme 1). The results are
summarized in Table 2.
As expected a small degree of H/D exchange is taking place at the most
acidic site a to the methylsulfinyl moiety. H/D exchange is however not
observed at the other sites. H/D exchange analysis of d8-1 in water at pH 7
over a period of 4 days using GC-MS did reveal a 65% H/D exchange at C-1
along with a conversion of the NCS functionality into its hydrated derivative.
The structure of the hydrated derivative is as follows:
Analytically pure d8-erucin could be obtained by a similar method omitting
the oxidation step. Erucin is the major isothiocyanate found in rocket salad
and shares the same biological activity as sulforaphane.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 501–507

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Conclusion
We have established a reliable and efficient synthesis of octadeutero-
sulforaphane and erucin. We will use these stable isotope labelled compounds
in biological studies to find answers to problems relating to their metabolism
and pharmocokinetics so as to ultimately be in a position to issue sound advice
to the consumer regarding brassica consumption and their potential health
benefits.
Acknowledgement
NK would like to thank the Association for International Cancer Research
(AICR) for financial support for this work.
References
1. Steinmetz KA. Cancer Causes Control 2000; 2: 325–357.
2. American Institute for Cancer Research. Dietary Phytochemicals in Cancer
Prevention and Treatment. Plenum Press: New York, 1996.
3. Cottaz S, Henrissat B, Driguez H. Biochemistry 1996; 35: 15256–15259.
4. Iori R, Rollin P, Streicher H, Thiem J, Palmieri S. FEBS Lett 1996; 385: 87–90.
5. Bones AM, Rossiter JT. Physiol Plant 1996; 97: 194–208.
6. Stoewsand GS. Food Chem Toxicol 1995; 33: 537–543.
7. Heaney RK, Fenwick GR. Identifying toxins and their effects: glucosinolates. In
Natural Toxicants in Food, Watson DH (ed.). vol. 1. Department of Health,
Ministry for Agriculture and Fisheries, London. 2000; 76–100.
8. Kuhnert N. Nachr Chem Tech 2001; 50: 142–147.
9. van Lieshout EM, Posner GH, Woodard BT, Peters WH. Biochim Biophys Acta
1998; 1379: 325–336.
10. Paolini M, Biagi GL, Cantelli-Forti G, Barcelo S, Gardiner JM, Gescher A,
Chipman JK. Carcinogenesis 1997; 18: 1435–1436.
11. Zhang Y, Kensler TW, Cho CG, Posner GH, Talalay P. Proc Natl Acad Sci USA
1994; 91: 3147–3150.
12. Rosa EAS, Heaney RK, Fenwick GR, Portas CAM. Hortic Rev 1996; 99–215.
13. Chiang WK, Pusateri DJ, Leitz RA. J Agric Food Chem 1998; 46: 1018–1021.
14. Fahey JW, Talalay P. Food Chem Toxicol 1999; 37: 973–979.
15. Gerhauser C, You M, Liu J, Moriarty RM, Hawthorne M, Mehta RG. Cancer
Res 1997; 57: 272–278.
16. Maheo K, Morel F, Langouet S, Kramer H, Le Ferrec E, Ketterer B. Cancer Res
1997; 57: 3649–3652.
17. Gamet-Payrastre L, Lumeau S, Gasc N, Cassar G, Rollin P, Tulliez J. Anticancer
Drugs 1998; 9: 141–148.
18. Heiss E, Herhaus C, Klimo K, Bartsch H, Gerhauser C. J Biol Chem 2001; 276:
32008–32015.
.
19. Schenk WA, Durr M. Chem Eur J 1997; 3: 713–716.
20. Karrer P, Schmid H. Helv Chim Acta 1948; 31: 1497–1507.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 501–507

SYNTHESIS OF 1, 10, 2, 20, 3, 30, 4, 40 -
507
21. Kuhnert N, Williamson G, Holst B. J Label Compd Radiopharm 2001; 44:
347–355.
22. Whitesell JK, Wong MS. J Org Chem 1994; 59: 597–599.
23. Furniss BS, Hannaford AJ, Smith PWG, Tatchell AR. Vogel’s Textbook of
Practical Organic Chemistry (5th ed). Longman Scientific & Technical: New
York, 1989.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 501–507