Syntheses of perdeuterated indoles and derivatives as probes for the biosyntheses of crucifer phytoalexins

Article abstract of DOI:10.1002/jlcr.1028

A simple two-step preparation of [²H₄]indole, a starting material necessary for the synthesis of various crucifer metabolites, starting with readily available ¹H NMR solvent [²H ₅]nitrobenzene (99% de

Full text of DOI:10.1002/jlcr.1028

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 33–45.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1028
Research Article
Syntheses of perdeuterated indoles and derivatives as
probes for the biosyntheses of crucifer phytoalexins
M. Soledade C. Pedras* and Denis P. O. Okinyo
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon,
SK S7N 5C9, Canada
Summary
A simple two-step preparation of [2H4]indole, a starting material necessary for the
1
synthesis of various crucifer metabolites, starting with readily available H NMR
solvent [2H5]nitrobenzene (99% deuterated) was developed. [4,5,6,7-2H4]Indole
99% deuterated at the specified positions was then used to synthesize [40,50,
60,70-2H4]indolyl-3-acetaldoxime,
[40,50,60,70-2H4]1-methoxyindolyl-3-acetaldoxime,
[100,100,100,40,50,60,70-2H7]1-methoxyindolyl-3-acetaldoxime, [40,50,60,70-2H4]1-methoxy-
brassinin, and [3,3,3,40,50,60,70-2H7]1-methoxybrassinin. Copyright # 2005 John
Wiley & Sons, Ltd.
Key Words: 1-methoxybrassinin; 1-methoxyindolyl-3-acetaldoxime; brassinin; indolyl-
3-acetaldoxime; tetradeuterated indole
Introduction
The interesting array of phytoalexins, i.e. chemical defenses, produced by
plants of the family Cruciferae (Brassicaceae) has prompted diverse studies
dealing with their syntheses, biosynthesis, biotransformation, and biological
activity.1 In particular, the correlation between phytoalexins and defense
pathways operating in plants2 explains both the interest in and significance of
the biosynthetic pathway of crucifer phytoalexins. Knowledge of such a
pathway(s) is of great importance in the genetic manipulation of secondary
metabolites of crucifers. Crucifer crops are the third largest source of vegetable
oils (canola, rapeseed, and mustard oils) and include as well a great variety of
vegetables used worldwide as staple food (broccoli, cauliflower, cabbage,
*Correspondence to: M. Soledade C. Pedras, Department of Chemistry, University of Saskatchewan, 110
Science Place, Sask., Canada SK S7N 5C9. E-mail: s.pedras@usask.ca
Contract/grant sponsor: Natural Sciences and Engineering Research Council of Canada
Contract/grant sponsor: University of Saskatchewan
Copyright # 2005 John Wiley & Sons, Ltd.
Received 30 September 2005
Revised 26 October 2005
Accepted 28 October 2005

34
M. S. C. PEDRAS, D. P. O. OKINYO
radish, rutabaga, turnip). Importantly, a number of epidemiological
studies suggest that cruciferous vegetables protect against cancer by
modulating carcinogen metabolism.3 Related studies attributed this modula-
tion to indole-containing metabolites4 such as 1-methoxyindole-3-carbinol,
which appears to show higher efficiency in the induction of cytochrome P450
hepatic enzymes.5
Crucifer phytoalexins,1 including camalexins,6 are biosynthetically
derived from tryptophan ð1Þ. Unambiguous biosynthetic studies carried
out independently by various groups have demonstrated that (S)-tryptophan
ð1Þ is the precursor of indolyl-3-acetaldoxime ð2Þ, which in turn is a pre-
cursor of the phytoalexins brassinin ð4Þ, a precursor of several other
phytoalexins ð6; 9; 10; 12215Þ, camalexin ð8Þ, and indolyl-3-acetonitrile ð11Þ,
and the indole glucosinolate glucobrassicin ð7Þ. Importantly, the C-2 of 13C-
labeled tryptophan ð1Þ was incorporated into the dithiocarbamic carbon
of brassinin ð4Þ, suggesting a Lossen-type rearrangement.7 Despite the
importance and number of naturally occurring compounds containing the
1-methoxyindole moiety, the first 1-methoxyindolyl biosynthetic inter-
mediate was established only very recently, using [2H3]1-methoxyindolyl-3-
acetaldoxime (3, R ¼¼ OC2H3, Scheme 1).8 In continuation of those
biosynthetic studies, it was crucial to develop synthetic routes to perdeutera-
ted indolyl-3-acetaldoximes 2a, 3a and 3b and brassinins 5a and 5b. Here
we report the syntheses and characterization of new perdeuterated
indoles in which the isotopic composition at the selected positions is
about 99%.
Scheme 1. Biosynthetic pathway of crucifer phytoalexins
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 33–45

SYNTHESES OF PERDEUTERATED INDOLES
35
Results and discussion
The use of perdeuterated compounds in biosynthetic studies is invaluable as
extremely small amounts of deuterated metabolites can be detected
unambiguously by mass spectrometry, e.g. ESI-HRMS or APCI-HRMS.9
The indole derivatives of immediate interest in biosynthetic studies of
phytoalexins need to contain at least three deuterium atoms, as this number
will allow the unambiguous detection and assignment of labeled phytoalexins
using HRMS analysis of m/z [M+3]+/ꢀ and corresponding fragment ions m/z
[MꢀX+3]+/ꢀ. The target compounds were selected containing different
deuterium composition: [40,50,60,70-2H4]indolyl-3-acetaldoxime ð2aÞ, [40,50,60,
70-2H4]1-methoxyindolyl-3-acetaldoxime ð3aÞ, [100,100,100,40,50,60,70-2H7]1-meth-
oxyindolyl-3-acetaldoxime ð3bÞ, [40,50,60,70-2H4]1-methoxybrassinin ð5aÞ, and
[3,3,3,40,50,60,70-2H7]1-methoxybrassinin ð5bÞ. The required starting material
for these syntheses, [4,5,6,7-2H4]indole ð17aÞ, needed to be at least 99%
deuterated; however, commercially available perdeuterated indoles either did
not offer this percentage of deuterium content at specific sites or were
extremely expensive. Subsequently, after consideration of a previous synthesis
of 99% tetradeuterated indole,10 which included the preparation of [2H4]2-
nitrophenylacetonitrile from [2H8]toluene,11 a rather difficult and somewhat
hazardous preparation, we searched for a shorter route to [2H4]2-nitrophe-
nylacetonitrile that could potentially provide access to [2,3-13C2]indole as well.
The well-known vicarious nucleophilic substitution of stabilized R-chlorocar-
banions with nitrobenzene12 appeared to provide a ready entry to various
isotopically labeled indoles. Thus, [2H4]2-nitrophenylacetonitrile was obtained
from [2H5]nitrobenzene and provided access to [4,5,6,7-2H4]indole ð17aÞ, from
which the readily accessible key intermediate [2H4]indolyl-3-acetonitrile ð11aÞ
could be prepared.13,14 The required deuterated oxime 2a could be readily
obtained from 11a,15 but the synthesis of perdeuterated Nb-acetyl-1-
methoxytryptamine ð19a; 19bÞ from which the corresponding oximes could
be obtained through oxidation, required a higher number of steps. [2H4]Indole
ð17aÞ was the starting material also used to prepare deuterated methoxyindole
ð21aÞ and methoxybrassinins 5a and 5b via [2H4]indole-3-carboxaldehyde
oxime ð22aÞ, using a well-established route for non-labeled materials.1
Subsequently, [4,5,6,7-2H4]indole ð17aÞ was obtained in two steps and 24%
overall yield, starting with the readily available 1H NMR solvent
[2,3,4,5,6-2H5]nitrobenzene (16a, 99%, 2H5) and 2-chloroacetonitrile,11 as
summarized in Scheme 2 and described in the Experimental section.
[40,50,60,70-2H4]Indolyl-3-acetonitrile ð11aÞ was prepared by reaction of
indolyl-magnesium iodide (prepared from [2H4]indole ð17aÞ, and Mg/methyl
iodide) with bromoacetonitrile.13,14 Reduction of indolyl-3-acetonitrile ð11aÞ
to indolyl-3-acetaldehyde employing DIBAL-H, followed by hydrolysis15
and treatment with HONH2 ꢁ HCl and NaOAc yielded the desired
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 33–45

36
M. S. C. PEDRAS, D. P. O. OKINYO
2
2
1
H
2
2
4'
3'
H
5'
N
OH
2'
N
H
6'
7'
H
2
2a
H
4. DIBAL-H
5. NH OH.HCl
2
NaOAc, EtOH
2
33%
CN
H
2
2
NO2
H
2
2
2
2
2
H
2
2
2
1
H
H
1. ClCH CN, DMSO, 40%
H
2
3. Mg, CH I
3
2. 10% Pd/C, H , EtOAc
2
61%
BrCH CN,
2
Et O, 57%
2
2
N
H
H
N
H
H
H
H
2
2
H
H
H
11a
17a
16a
6. 10% Pd/C, H
2
Ac O
2
7. NaBH CN,
3
AcOH
NHAc
2
2
1
H
4'
2
NHAc
1
2
H
2
H
2
9. NaOH, MeOH,
reflux
H
5'
8. Na WO , H O
2 2
2
2
4
N
OH
2
2
H
H
1'
N
2
(C H O) SO ,
2
3
2
2
6'
10. Na WO ,
H
2
4
2
2
N
7'
N
H
K CO , MeOH
OC H
H
2
2
3
3
H O , MeOH
H
H
2
2
2
3b
2
OC H
1"
2
3
18a
H
H
4%
19b
11. Na WO , H O
2 2
2
4
(CH O) SO ,
3
2
2
K CO , MeOH
2
3
2
2
NHAc
1
2
2
1
3
H
2
H
2
2
12. NaOH, MeOH,
reflux
2
H
H
N
OH
2'
1'
N
2
13. Na WO ,
N
H
2
4
H
OCH
OCH
3
H O , MeOH, 5%
2
2
2
H
H
3a
19a
Scheme 2. Synthesis of perdeuterated indolyl-3-acetaldoximes 2a, 3a and 3b
[40,50,60,70-2H4]indolyl-3-acetaldoxime ð2aÞ in acceptable yields. [2H7]Nb-acetyl
1-methoxytryptamine ð19bÞ was prepared from 18a after reduction with
NaBH3CN, followed by oxidation with Na2WO4/H2O2,16 and methylation
of the 1-hydroxyindolyl intermediate with (CD3O)2SO2. [2H4]Nb-acetyl
1-methoxytryptamine ð19aÞ was prepared similar to the [2H7] compound 19b
but using ðCH3OÞ2SO2,16 instead of the hexadeuterated methylating reagent.
Perdeuterated 1-methoxyindolyl-3-acetaldoximes 3a and 3b were then
obtained by hydrolysis of the Nb-acetyl group followed by oxidation of the
amine to oxime using Na2WO4/H2O2.17 This oxidation step proved rather
difficult, mostly due to decomposition of the product under the reaction
conditions used, however no simple and efficient methods were found to
oxidize aliphatic amines to oximes. For example, CH3ReO3/H2O2 catalyzed
oxidation of primary alkylamines possessing the a-C–H bond was found to
yield mixtures of oximes, nitroso dimers, and azoxy compounds.18
Next, perdeuterated 1-methoxybrassinins 5a and 5b were obtained from
[4,5,6,7-2H4]indole as shown in Scheme 3. First, reduction of [2H4]indole ð17aÞ
with NaBH3CN in AcOH followed by oxidation with Na2WO4/H2O2 and
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 33–45

SYNTHESES OF PERDEUTERATED INDOLES
37
2
2
2
H
2
H
H
1
H
N
OH
3. POCl , DMF
3
93%
2
2
2
2
4
3
H
2
2
H
4'
H
3'
H
1. NaBH CN
3
2. Na WO , H O
2 2
2
4
5
5'
6'
2'
1
2
AcOH, 82%
4. NH OH.HCl
2
N
(CH O) SO , K CO
N
H
N
H
2
3
2
2
2
3
2
6
H
N
H
H
H
OCH
7
3
7'
Na CO ,
2
OCH
2
2
2
3
2
3
MeOH, 52%
H
2
H
H
H
EtOH, 99%
20a
21a
17a
22a
2
2
1
H
3
5. NaBH CN, TiCl
3 3
H
H
NH
NH
3
6. Py, Et N, CS ,
2
3
2
2
4'
SC H
3'
1'
2
2
3
H
NH OAc, MeOH
H
4
2
60 min, 0 °C
5'
2'
S
3
2
2
N
7. IC H , 68%
3
N
H
6'
7'
H
OCH
OCH
2
2
H
5b
23a
6. Py, Et N, CS ,
3
2
60 min, 0 °C
7. ICH , 67%
3
2
1
H
3
SCH
NH
2
2
4'
3'
3
H
2
5'
2'
S
1'
N
6'
H
7'
OCH
3
2
H
5a
Scheme 3. Synthesis of perdeuterated 1-methoxybrassinins 5a and 5b
16
methylation with ðCH3OÞ2SO2
yielded [2H4]1-methoxyindole ð21aÞ in
moderate yield. Standard Vilsmeier-Haack conditions yielded [2H4]1-methox-
yindole-3-carboxaldehyde, which was first treated with HONH2 ꢁ HCl and
NaOAc followed by reduction of the resulting oxime with NaBH3CN, TiCl3,
and NH4OAc in MeOH to yield [40,50,60,70-2H4]1-methoxyindolyl-3-methana-
mine ð23aÞ.19 The synthesis of [2H4] and [2H7]1-methoxybrassinins 5a and 5b,
followed previously set routes,1 which afforded the desired compounds in
reasonable yields.
All the synthesis described above afforded perdeuterated products 99%
labeled at the indicated sites.
Experimental
General
All chemicals were purchased from Sigma-Aldrich Canada Ltd., Oakville,
Ont., except for deuterated solvents that were purchased from Cambridge
Isotopes Laboratories Inc., Andover, MA. All solvents were high-performance
liquid chromatograph (HPLC) grade and used as such, except CHCl3 and
CH2Cl2 that were redistilled. Preparative TLC: (Merck, Kieselgel 60 F254
)
20 ꢂ 20 cm ꢂ 0.25 mm; analytical TLC (Merck, Kieselgel 60 F254, aluminum
sheets) 5 ꢂ 2.5 cm ꢂ 0.2 mm; compounds were visualized by exposure to UV
and by dipping the plates in a 5% aqueous (w/v) phosphomolybdic acid
solution containing a trace of ceric sulfate and 4% (v/v) H2SO4, followed by
heating at 2008C. Flash column chromatography (FCC): silica gel Merck,
grade 60, mesh size 230–400, 60 A. HPLC analysis was carried out with an
Copyright # 2005 John Wiley & Sons, Ltd.
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38
M. S. C. PEDRAS, D. P. O. OKINYO
HPLC equipped with quaternary pump, automatic injector and diode array
detector (wavelength range 190–600 nm), degasser and a Hypersil ODS
column (5 mm particle size silica, 4.6 i.d. ꢂ 200 mm) equipped with an in-line
filter. HPLC mobile phase H2O–CH3CN: 75%/25%–0%/100%, for 45 min,
linear gradient and flow rate 1.0 ml/min. NMR spectra were recorded on
Bruker 500 MHz Avance spectrometers; for 1H (500 MHz), values were
referenced to CHCl3 (7.27 ppm) or CHD2CN (1.94 ppm), for 13C (125.8 MHz)
referenced to CHCl3 (77.2 ppm) or CD3CN (118.7 ppm). Mass spectra (MS)
were obtained on a VG 70 SE mass spectrometer (high resolution (HR),
electron impact (EI)), employing a solids probe.
[40,50,60,70-2H4]Indolyl-3-acetaldoxime (2a)
A solution of DIBAL-H (850 ml, 1.28 mmol) in toluene was added drop wise to
a solution of [40,50,60,70-2H4]indolyl-3-acetonitrile (100 mg, 0.63 mmol) in dry
toluene (8.0 ml) cooled to ꢀ788C under atmosphere of argon. The reaction
mixture was allowed to stir at ꢀ788C for 10 min, was diluted with ice-cold HCl
(10.0 ml, 2 M) and immediately extracted in EtOAc (20.0 ml ꢂ 3).15 The
combined organic extract was washed with H2O (10.0 ml ꢂ 2), was dried over
Na2SO4, and was concentrated under reduced pressure to yield crude
[40,50,60,70-2H4]indolyl-3-acetaldehyde, which was used for the next step
without purification. A solution of HONH2 ꢁ HCl (111 mg, 1.60 mmol) and
CH3COONa (131 mg, 1.60 mmol) in water (1.0 ml) was added to a cooled
solution (18C) of crude [40,50,60,70-2H4]indolyl-3-acetaldehyde in EtOH
(8.5 ml). The reaction mixture was allowed to stir at 18C for 10 min, and
then at r.t. for 15 min. The reaction mixture was concentrated, the residue was
dissolved in H2O (15.0 ml), extracted with EtOAc (15.0 ml ꢂ 4), the combined
organic extract was dried over Na2SO4 and was concentrated under reduced
pressure. Separation by FCC (CH2Cl2–MeOH, 97:3, v/v) yielded
[40,50,60,70-2H4]indolyl-3-acetaldoxime (2a, 36.8 mg, 33% yield from indolyl-
3-acetonitrile ð11aÞ).
1H NMR (500 MHz CD3CN): (two isomers, 1.1:0.9) major isomer: d 3.60
(d, J ¼ 6 Hz, H2-2), 7.12 (d, J ¼ 2 Hz, H-20), 7.49 (t, J ¼ 6 Hz, H-1), 8.37
(s, OH), 9.17 (br s, NH); minor isomer: d 3.78 (d, J ¼ 5 Hz, H2-2), 6.83
(t, J ¼ 5 Hz, H-1), 7.15 (d, J ¼ 1 Hz, H-20), 8.85 (s, OH), 9.17 (br s, NH).
HREIMS m/z measured 178.1044 (178.1044 calculated for C10H6D4N2O);
EIMS m/z (relative abundance) 178 [M]+ (76), 134 (100).
[40,50,60,70-2H4]1-Methoxyindolyl-3-acetaldoxime (3a)
Ten percent Pd/C (80 mg) was added to a solution of [40,50,60,70-2H4]indolyl-3-
acetonitrile (80 mg, 0.50 mmol) in Ac2O (4.0 ml), and the mixture was stirred at
r.t. under H2 atmosphere (balloon pressure). After 14 h the catalyst was filtered
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESES OF PERDEUTERATED INDOLES
39
off, the filtrate was diluted with toluene and concentrated under reduced
pressure, the residue was dissolved in CH2Cl2 (15.0 ml), was washed with a
10% solution of NaHCO3 (8.0 ml), H2O (8.0 ml ꢂ 2), and dried over Na2SO4.
After removal of the solvent under reduced pressure, crude [40,50,60,70-2H4]Nb-
acetyltryptamine was obtained (87.9 mg, 85%) in sufficient purity to use
directly in the next step. To a solution of [40,50,60,70-2H4]Nb-acetyltryptamine
(100 mg, 0.495 mmol) in glacial acetic acid (2.0 ml) at r.t., NaBH3CN (47 mg,
0.74 mmol) was added in portions through a canula. The reaction mixture was
allowed to stir for 3 h at room temperature, was diluted with H2O (5.0 ml), was
basified with NaOH, and was extracted with Et2O (15.0 ml ꢂ 3). The combined
organic extract was dried over Na2SO4 and concentrated under reduced
pressure to yield crude [40,50,60,70-2H4]Nb-acetyl-2,3-dihydrotryptamine
(117 mg, 99%), which was used in the next step without purification. A
solution of Na2WO4 ꢁ 2H2O (17.2 mg, 0.0943 mmol) in H2O (199 ml) was added
to a solution of [40,50,60,70-2H4]Nb-acetyl-2,3-dihydrotryptamine (112.8 mg,
0.542 mmol) in MeOH (2.2 ml) cooled to ꢀ208C under stirring, followed by
drop wise addition of a solution of H2O2 (520 ml, 5.42 mmol) in MeOH
(540 ml). After being stirred at r.t. for 10 min, K2CO3 (601 mg, 4.44 mmol) and
(CH3O)2SO2 (78 ml, 0.87 mmol) were added under vigorous stirring at r.t.16
After 60 min, the reaction mixture was diluted with H2O (12.0 ml), was
extracted in Et2O (20.0 ml ꢂ 3), the combined organic extract was dried over
Na2SO4 and was concentrated under reduced pressure to yield crude
[40,50,60,70-2H4]Nb-acetyl-1-methoxytryptamine (19a, 79.4 mg, 62%). Crude
[40,50,60,70-2H4]Nb-acetyl-1-methoxytryptamine in (79.4 mg) was dissolved in
15% methanolic solution of NaOH (20.0 ml) and the mixture was allowed to
reflux for 24 h. After removing the solvent under reduced pressure, the residue
was dissolved in water, was extracted with CH2Cl2–MeOH (95:5, v/v), was
dried over Na2SO4 and was concentrated under reduced pressure to yield
crude [40,50,60,70-2H4]1-methoxytryptamine (41 mg, 63%). An aqueous solution
of Na2WO4 ꢁ 2H2O (1.4 mg, 0.0042 mmol) in H2O (60 ml) was added to the
solution of [40,50,60,70-2H4]1-methoxytryptamine (41 mg, 0.21 mmol) in MeOH
(400 ml), the mixture was cooled to ꢀ158C and H2O2 (48 ml, 0.5 mmol) was
added under stirring.17 After being stirred for 60 min at r.t., the mixture was
diluted with H2O (5.0 ml), was basified with NaOH, was extracted with
CH2Cl2 (20.0 ml ꢂ 3), the combined organic extract was dried over Na2SO4
and the solvent was removed under reduced pressure to afford a crude residue
(24.3 mg). The crude residue was separated by preparative TLC (CH2Cl2–
MeOH, 95:5, v/v) to yield [40,50,60,70-2H4]1-methoxyindolyl-3-acetaldoxime
(3a, 4.8 mg, 5% over five steps).
1H NMR (500 MHz CDCl3): (two isomers, 1.1:0.9) major isomer d 3.64
(d, J ¼ 6 Hz, H2-2), 4.08 (s, OCH3), 7.15 (s, H-20), 7.60 (t, J ¼ 6 Hz, H-1);
%
minor isomer: d 3.83 (d, J ¼ 5 Hz, H2-2), 4.09 (s, OCH3), 6.93 (t, J ¼ 5 Hz,
%
Copyright # 2005 John Wiley & Sons, Ltd.
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40
M. S. C. PEDRAS, D. P. O. OKINYO
H-1), 7.17 (s, H-20). HREIMS m/z (relative abundance) measured 208.1142
(208.1149 calculated for C11H8D4N2O2); EIMS m/z (relative abundance) 208
[M]+ (100), 190 (25), 164 (45), 159 (54), 132 (70).
[100,100,100,40,50,70-2H7]1-Methoxyindolyl-3-acetaldoxime (3b)
A solution of Na2WO4 ꢁ 2H2O (32.3 mg, 0.098 mmol) in H2O (206 ml) was
added to a solution of [40,50,60,70-2H4]Nb-acetyl-2,3-dihydrotryptamine
(117 mg, 0.563 mmol) in MeOH (2.3 ml) under stirring.16 The mixture was
cooled to ꢀ208C and a solution of H2O2 (541 ml, 5.63 mmol) in MeOH (562 ml)
was added drop wise. After being stirred for 10 min at r.t., K2CO3 (623 mg,
4.50 mmol) and (C2H3O)2SO2 (85 ml, 0.90 mmol) were added to the reaction
mixture under vigorous stirring. After being stirred for 60 min at r.t., the
reaction mixture was diluted with H2O (12.0 ml), was extracted with Et2O
(20.0 ml ꢂ 3), the combined organic extract was dried over Na2SO4 and was
concentrated under reduced pressure to yield crude [100,100,100,40,50,60,70-2H7]Nb-
acetyl-1-methoxytryptamine (84.8 mg, 63%). Crude [100,100,100,40,50,60,
70-2H7]Nb-acetyl-1-methoxytryptamine (84.8 mg) was treated as reported
above for [40,50,60,70-2H4]Nb-acetyl-1-methoxytryptamine ð19aÞ to yield
[100,100,100,40,50,60,70-2H7]1-methoxyindolyl-3-acetaldoxime (3b, 4.2 mg, 4% over
five steps).
1H NMR (500 MHz CDCl3): (two isomers, 1.1:0.9) major isomer d 3.64
(d, J ¼ 6 Hz, H2-2), 7.15 (s, H-20), 7.60 (t, J ¼ 6 Hz, H-1); minor isomer:
d 3.83 (d, J ¼ 5 Hz, H2-2), 6.93 (t, J ¼ 5 Hz, H-1), 7.17 (s, H-20). HREIMS
m/z (relative abundance) measured 211.1335 (211.1338 calculated for
C11H5D7N2O2); EIMS m/z (relative abundance) 211 [M]+ (100), 193 (29),
167 (48), 159 (51), 132 (61).
[40,50,60,70-2H4]1-Methoxybrassinin (5a)
NaBH3CN (195 mg, 1.68 mmol) was added to a solution of [4,5,6,7-2H4]indole
(17a, 135 mg, 1.12 mmol) in glacial acetic acid (2.0 ml) under an argon
atmosphere. The reaction mixture was stirred at r.t. for 60 min, was diluted
with H2O (4.0 ml), was basified with NaOH, was extracted with Et2O
(30.0 ml ꢂ 3), the combined organic extract was dried over Na2SO4 and was
concentrated under reduced pressure. The crude material was separated by
FCC (CH2Cl2) to yield [4,5,6,7-2H4]indoline (20a, 113.2 mg, 82%). A solution
of Na2WO4 ꢁ 2H2O (53 mg, 0.16 mmol) in H2O (0.35 ml) was added to the
stirred solution of [4,5,6,7-2H4]indoline (113.2 mg, 0.920 mmol) in MeOH
(3.0 ml) and the suspension was then cooled to ꢀ208C. A solution of H2O2
(766 ml, 7.96 mmol) in MeOH (1.0 ml) was added drop wise to the cooled
suspension, the reaction mixture was stirred at r.t. for 10 min, after which solid
K2CO3 (1.02 g, 7.36 mmol) and (CH3O)2SO2 (133 ml, 1.47 mmol) were added
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SYNTHESES OF PERDEUTERATED INDOLES
41
under vigorous stirring.16 After being stirred for 10 min, the reaction mixture
was diluted with H2O (20.0 ml), was extracted with Et2O (30.0 ml ꢂ 3), the
combined organic extract was dried over Na2SO4 and was concentrated under
reduced pressure. The crude material was separated by FCC (hexane–CH2Cl2,
70:30, v/v) to yield [4,5,6,7-2H4]1-methoxyindole (21a, 73 mg, 52%). Freshly
distilled POCl3 (69 ml, 0.75 mmol) was added to a solution of [4,5,6,7-2H4]1-
methoxyindole (102 mg, 0.68 mmol) in DMF (0.6 ml) and the reaction mixture
was stirred at r.t. After 40 min, a solution of aqueous NH3 (4.0 ml, 28%) was
added to the reaction mixture, the reaction mixture was extracted with Et2O
(10.0 ml ꢂ 3), the combined organic extract was dried over Na2SO4 and was
concentrated under reduced pressure to yield [40,50,60,70-2H4]1-methoxyindole-
3-carboxaldehyde (113 mg, 93%). A solution of HONH2 ꢁ HCl (80.6 mg,
1.16 mmol) and Na2CO3 (61.5 mg, 0.580 mmol) in H2O (810 ml) was added
to a solution of [40,50,60,70-2H4]1-methoxyindole-3-carboxaldehyde (103 mg,
0.580 mmol) in EtOH (2.7 ml). The resulting reaction mixture was refluxed at
858C for 60 min, was diluted with H2O (3.0 ml) and was extracted with
Et2O (20.0 ml ꢂ 3). The combined organic extract was washed with brine
(10.0 ml ꢂ 2), was dried over Na2SO4 and was concentrated under reduced
pressure to yield [40,50,60,70-2H4]1-methoxyindole-3-carboxaldehyde oxime
(22a, 122.1 mg, 99%). NaBH3CN (163 mg, 2.58 mmol) and NH4OAc
(219 mg, 2.84 mmol) were added to a solution of [40,50,60,70-2H4]1-methox-
yindole-3-carboxaldehyde oxime (50 mg, 0.26 mmol) in MeOH (640 ml) cooled
to 08C and the resulting mixture was treated with TiCl3 in 2 M HCl (795 ml,
2.04 mmol) neutralized with NaOH.19 After being stirred for 15 min at 08C, the
reaction mixture was diluted with H2O (2.2 ml), was basified with NaOH and
was extracted with CH2Cl2 (15.0 ml ꢂ 3). The combined organic extract was
dried over Na2SO4 and was concentrated under reduced pressure to yield
crude [40,50,60,70-2H4]1-methoxyindolyl-3-methanamine (23a, 40.3 mg) which
was used in the next step without purification. Et3N (34 ml, 0.25 mmol) and
CS2 (13 ml, 0.22 mmol) were added to a solution of [40,50,60,70-2H4]1-
methoxyindolyl-3-methanamine (40.3 mg, 0.224 mmol) in pyridine (105 ml)
cooled to 08C. After being stirred for 60 min at 08C, CH3I (14 ml, 0.22 mmol)
was added to the reaction mixture which was kept at 58C for 90 min. The
reaction mixture was diluted with H2O (2.0 ml), was extracted with Et2O
(10.0 ml ꢂ 2), the combined organic extract was dried over Na2SO4 and was
concentrated under reduced pressure. The reaction mixture was separated
by FCC (hexane–CH2Cl2) to yield [40,50,60,70-2H4]1-methoxybrassinin (5a,
46.5 mg, 67% from [40,50,60,70-2H4]1-methoxyindole-3-carboxaldehyde oxime
ð22aÞÞ.
1H NMR (500 MHz CDCl3): d 2.67 (s, SCH3), 4.12 (s, OCH3), 5.04
%
%
(d, J ¼ 4:5 Hz, H2-1), 7.04 (br s, NH), 7.35 (s, H-20). HREIMS m/z (relative
abundance) measured 270.0794 (270.0795 calculated for C12H10D4N2OS2);
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M. S. C. PEDRAS, D. P. O. OKINYO
EIMS m/z (relative abundance), 270 [M]+ (6), 239 (64), 222 (15), 191 (8), 164
(100), 149 (15), 133 (43).
[3,3,3,40,50,60,70-2H7]1-Methoxybrassinin (5b)
NaBH3CN (179 mg, 2.84 mmol) and NH4OAc (241 mg, 3.13 mmol) were
added to a solution of [40,50,60,70-2H4]1-methoxyindole-3-carboxaldehyde
oxime (22a, 55 mg, 0.284 mmol) in MeOH (700 ml) cooled to 08C. The
resulting reaction mixture was treated with a solution of TiCl3 in 2 M HCl
(876 ml, 2.25 mmol) neutralized with NaOH.19 After being stirred for 15 min at
08C, the reaction mixture was diluted with H2O (2.4 ml), was basified with
NaOH and was extracted with CH2Cl2 (15.0 ml ꢂ 3). The combined organic
extract was dried over Na2SO4 and was concentrated under reduced pressure
to yield [40,50,60,70-2H4]1-methoxyindolyl-3-methanamine (23a, 44 mg) which
was used in the next step without purification. Et3N (37 ml, 0.27 mmol) and
CS2 (15 ml, 0.24 mmol) were added to a solution of [40,50,60,70-2H4]1-
methoxyindolyl-3-methanamine (44 mg, 0.24 mmol) in pyridine (118 ml) cooled
to 08C. After being stirred for 60 min at 08C, CD3I (16 ml, 0.244 mmol) was
added and the reaction mixture was kept at 58C for 90 min. The reaction
mixture was diluted in H2O (2.0 ml), was extracted with Et2O (10.0 ml ꢂ 2), the
combined organic extract was dried over Na2SO4 and was concentrated under
reduced pressure. The reaction mixture was separated by FCC (hexane–
CH2Cl2) to yield [3,3,3,40,50,60,70-2H7]1-methoxybrassinin (5b, 52.7 mg, 68%
yield from [40,50,60,70-2H4]1-methoxyindole-3-carboxaldehyde oxime ð22aÞÞ.
1H NMR (500 MHz CDCl3): d 4.11 (s, ðOCH3Þ, 5.02 (br s, H2-1), 7.04 (br s,
%
NH), 7.32 (br s, H-20). HREIMS m/z (relative abundance) measured 273.0984
(273.0987 calculated for C12H7D7N2OS2); EIMS m/z (relative abundance) 273
[M]+ (7), 242 (100), 191 (12), 164 (85), 149 (19), 133 (40).
[40,50,60,70-2H4]Indolyl-3-acetonitrile (11a)
CH3I (400 ml, 2.80 mmol) was added to Mg (turnings, 80 mg, 3.3 mmol) in dry
Et2O (5.0 ml) with stirring, under an argon atmosphere. After the Mg was
consumed, excess CH3I was distilled off and fresh dry Et2O (4.0 ml) was added.
A solution of [4,5,6,7-2H4]indole (100 mg, 0.830 mmol) in dry Et2O (700 ml)
was added drop wise and the mixture was allowed to stir for 15 min at r.t.,
followed by drop wise addition of bromoacetonitrile (160 ml, 2.70 mmol).13,14
After being stirred for 20 min, the reaction mixture was concentrated under
reduced pressure and the residue was dissolved in water H2O (40.0 ml). The
resulting mixture was neutralized with HCl (2.0 ml, 1 M) and was extracted
with CH2Cl2 (30.0 ml ꢂ 3). The combined organic extract was dried over
Na2SO4 and was concentrated to dryness under reduced pressure.
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SYNTHESES OF PERDEUTERATED INDOLES
43
Separation by FCC (hexane–CH2Cl2, 1:1–1:4) yielded [40,50,60,70-2H4]indolyl-3-
acetonitrile (11a, 75.7 mg, 57% yield).
1H NMR (500 MHz CDCl3): d 3.86 (s, H2-2), 7.24 (t, J ¼ 1 Hz, H-20), 8.23
(br s, NH). HREIMS m/z (relative abundance) measured 160.0940 (160.0939
calculated for C10H4D4N2); EIMS m/z (relative abundance) 160 [M]+ (62),
159 (100), 134 (32).
[4,5,6,7-2H4]Indole (17a)
A solution of [2,3,4,5,6,-2H5]nitrobenzene (16a, 4.20 ml, 39.4 mmol) and
chloroacetonitrile (250 ml, 3.94 mmol) in DMSO (3.9 ml) was added drop wise
over 30 min to a thoroughly stirred suspension of NaOH (1.60 g, 39.4 mmol) in
DMSO (3.9 ml) at 208C.12 After 60 min, the reaction mixture was poured into
ice-cold HCl (25.0 ml, 1 M), was diluted with H2O (10.0 ml), and was extracted
with CHCl3 (50.0 ml ꢂ 3). The combined organic extract was washed with H2O
(70.0 ml), was dried over Na2SO4 and was concentrated under reduced
pressure. Separation by FCC (hexane–CH2Cl2, 100:0–25:75) yielded a mixture
of [3,4,5,6,-2H4]2-nitrophenylacetonitrile and [2,3,5,6,-2H4]4-nitrophenylace-
tonitrile (in a 10:1 ratio, 40% yield). Next, 10% Pd on C (220 mg) was added
to the solution of [2H4]2- and [2H4]4-nitrophenylacetonitriles (200 mg) in
EtOAc (20.0 ml) and the resulting reaction mixture was stirred under H2
atmosphere (balloon pressure) at r.t. After 40 h, the catalyst was filtered off,
and the filtrate was concentrated under reduced pressure. The residue was
taken in CH2Cl2 (15.0 ml), was washed with HCl (10.0 ml ꢂ 2, 1 M), was dried
over Na2SO4 and was concentrated under reduced pressure to yield
[4,5,6,7-2H4]indole (17a, 97.7 mg, 61% yield).
1H NMR (500 MHz CDCl3): d 6.61 (br s, H-3), 7.23 (br s, H-2), 8.12 (br s,
NH). HREIMS m/z (relative abundance) measured 121.0827 (121.0830
calculated for C8H3D4N); EIMS m/z (relative abundance) 121 [M]+ (100),
94 (22), 71 (11), 69 (12), 57 (17).
Conclusion
To the best of our knowledge, only one synthesis of perdeuterated indole ð17aÞ
and indole containing phytoalexins with 99% incorporation at multiple sites
has been reported to date;10 however, this new route to indole starting with
[2H5]nitrobenzene ð16aÞ is simpler, less hazardous and more efficient,
providing an accessible method for the synthesis of perdeuterated brassinins
ð5a; 5bÞ. Furthermore, a simple route to perdeuterated indolyl-3-acetonitrile
ð11aÞ, a key intermediate for the preparation of perdeuterated indolyl-3-
acetaldoxime 2a and 1-methoxyindolyl-3-acetaldoximes 3a and 3b is described
for the first time. The availability of 99% labeled indole derivatives 2a, 3a, 3b,
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44
M. S. C. PEDRAS, D. P. O. OKINYO
5a, 5b and 11a will facilitate probing the various biosynthetic relationships
among crucifer phytoalexins using LC-HRMS techniques. As well, consider-
ing that indolyl-3-acetaldoxime ð2Þ is an intermediate in other important
metabolic pathways of Brassicaceae, such as those of plant hormones (auxins)
and indole glucosinolates,20 availability of synthetic methodology to make
these perdeuterated compounds will have additional applications.
Acknowledgements
Support for the authors’ work was obtained in part from the Natural Sciences
and Engineering Research Council of Canada (Discovery grant to MSCP) and
the University of Saskatchewan (Graduate Teaching Assistantship to DPOO).
We would like to thank K. Brown and K. Thoms, Department of Chemistry,
University of Saskatchewan, for technical assistance to obtain NMR and MS
data, respectively.
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