The preparation of side chain functionalized analogues of coenzyme Q for protein conjugation studies

Article abstract of DOI:10.1039/b407659a

The synthesis of two analogues of CoQ (10 and 13) suitable for conjugation to a peptide or protein, and hence the development of an ELISA immunoassay, is presented. These analogues were synthesized from the protected quinone, 1-bromo-2-methyl-3,4,5,6-tetramethoxybenzene (1), itself prepared from commercially available CoQ-0 (3). Model coupling studies of one of the analogues (10) to N-acetyl-L-lysine methyl ester and a lysine containing dipeptide (N-acetylglycine-L-lysine methyl ester) were also undertaken as a first step to monoclonial antibody production.

Full text of DOI:10.1039/b407659a

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A R T I C L E
The preparation of side chain functionalized analogues of
coenzyme Q for protein conjugation studies
Alison M. Daines and Andrew D. Abell*
Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch,
New Zealand. E-mail: a.abell@chem.canterbury.ac.nz
Received 25th May 2004, Accepted 24th June 2004
First published as an Advance Article on the web 27th July 2004
The synthesis of two analogues of CoQ (10 and 13) suitable for conjugation to a peptide or protein, and hence the
development of an ELISA immunoassay, is presented. These analogues were synthesized from the protected quinone,
1-bromo-2-methyl-3,4,5,6-tetramethoxybenzene (1), itself prepared from commercially available CoQ-0 (3). Model
coupling studies of one of the analogues (10) to N-acetyl-L-lysine methyl ester and a lysine containing dipeptide (N-acetyl-
glycine-L-lysine methyl ester) were also undertaken as a first step to monoclonial antibody production.
carboxyl group at the side-chain terminus suitable for conjugation
Introduction
to a peptide or protein. In addition, analogue 10 was coupled to
The coenzyme Qn family, also known as ubiquinones (Fig. 1), have
N-acetyl-L-lysine methyl ester and a lysine containing dipeptide (N-
a number of important biofunctions that include acting as mobile
acetyl-glycine-lysine methyl ester) to demonstrate the feasibility of
mediators for electron transfer and protein translocation between
its use in protein conjugation studies that would form a basis for
redox enzymes in the electron transport chain of mitochondria
monoclonal antibody production and hence the development of an
and bacterial respiratory systems.1–3 Coenzyme Q is also known
ELISA based detection method.
to act as an antioxidant by reducing free radicals that can cause
damage to structural lipids or proteins in the membrane, not only
Results and discussion
in mitochondria but in any cellular CoQ containing membranes.3 In
addition, a deficiency of CoQ has been observed in many diseased
states, and supplementation with exogenous CoQ is thought to be
beneficial to some disorders including cancer,4–6 diabetes,7,8 heart
disease9,10 and neurodegenerative disease.11,12 For these reasons it is
important to have efficient methods for the preparation and detec-
tion of these natural products and derivatives thereof.
Our synthesis of 1 involved simple protection of 2,3-dimethoxy-
5-methyl-1,4-benzoquinone (CoQ-0) (3) followed by aryl bromi-
nation as detailed in Scheme 1. In particular, reaction of 3 with
stannous chloride and concentrated hydrochloric acid afforded the
hydroquinone 4, which was not purified. This crude material was
suspended in dimethylsulfate, potassium hydroxide was added in
aliquots, and the mixture heated at 80 °C for one hour to give 2 in
85% over two steps. Finally, 2 was treated with bromine for 10 min
to give 1-bromo-2-methyl-3,4,5,6-tetramethoxybenzene (1) in 84%
yield. This simple three step protocol affords 1 in 71% overall yield
from the commercially available starting material 3. Importantly,
the products do not require purification by chromatography and as
such the methodology is suitable for large scale synthesis.
Fig. 1 Coenzyme Q and some important chemical intermediates.
The protected quinone 1-bromo-2-methyl-3,4,5,6-tetramethoxy-
benzene (1), a key starting material for the synthesis of coenzyme
Q and its analogues, is conveniently derived from 1-methyl-2,3,4,5-
tetramethoxybenzene (2).13 However, published syntheses of 2 are
time consuming, complex and low yielding. A review of the litera-
ture shows that a number of groups have synthesised 2 via com-
plicated multistep procedures starting from highly functionalised
starting materials such as glucose,14 and pyrogallol.13 Keinan and
Eren15 published a two step synthesis of 2 from p-cresol in 71%
yield, but in our hands this procedure gave a complex, intractable
mixture from which only a very low yield of the desired derivative
2 was isolated. There are also limited reports on the preparation
of various analogues of coenzyme Q including fluorescently16 and
isotopically labelled analogues,17 O-alkylated derivatives,18,19 and
analogues with side chain modifications.20–23 These compounds
were synthesised via a series of functional group interconversions
of CoQ compounds with varying chain length.
Scheme 1 i. SnCl2, c.HCl, EtOH; ii. dimethylsulfate, KOH, 80 °C, 85%
(two steps); iii. Br2, CH2Cl2, 5 °C, 84%.
1-Bromo-2-methyl-3,4,5,6-tetramethoxybenzene (1) was then
used to prepare aldehyde 8, a key precursor to side-chain func-
tionalised analogues (10 and 13) of coenzyme Q, Schemes 2 and
3. The aryl bromide 1 was treated with nBuLi and copper bromide
dimethylsulfide complex to give the corresponding diaryl cuprate
which was coupled with geranyl bromide13 to give 5 in an unopti-
mised yield of 48%. Treatment of 5 with N-bromosuccinimide then
gave the bromohydrin (6), which upon reaction with potassium
carbonate gave the corresponding epoxide (7). Cleavage of the
epoxide (7) with periodic acid gave the desired aldehyde (8), which
was used subsequently without purification.
With the key aldehyde 8 in hand we set about the preparation
of side chain functionalised analogues of coenzyme Q, 10 and 13,
Scheme 3. Analogue 10 was synthesised in two steps; the aldehyde
8 was oxidized with silver nitrate and sodium hydroxide in ethanol
to the carboxylic acid 9, the quinone core of which was deprotected
In this paper we present an efficient and reproducible three step
synthesis of 1 starting from commercially available 2,3-dimethoxy-
5-methyl-1,4-benzoquinone (CoQ-0). We also report the prepara-
tion of analogues of coenzyme Q (10 and 13), from 1, that bear a
T h i s j o u r n a l i s
©
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 3 7 1 – 2 3 7 5
2 3 7 1

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acid of the analogue using standard peptide coupling conditions. It
is important to note that lysine residues are commonly located on
the surface of a protein so coupling to lysine is desirable for protein
coupling experiments. The analogue 10 was used in these studies
and its reaction with N--acteyl-lysine methyl ester and N-acetyl-
glycine-lysine methyl ester proceeded cleanly in the presence of
EDCI, HOBt and DIPEA in dichloromethane to give 14 and 15 in
63% and 34% yield, respectively (see Scheme 4).
Scheme 2 i. nBuLi, CuBr·Me2S, geranyl bromide, Et2O, 0 °C–R.T., 48%;
ii. NBS, THF/H2O, 65%; iii. K2CO3, MeOH, 78%; iv. periodic acid, THF,
Et2O, 85%.
Scheme 4 i. EDCI, HOBt, DIPEA, CH2Cl2.
with cerric ammonium nitrate to give 10 (Scheme 3). Analogue 13
was prepared from aldehyde 8 as shown in Scheme 3; reduction
of the aldehyde with sodium borohydride gave the corresponding
alcohol (11), which was reacted with succinic anhydride in the pres-
ence of triethylamine and DMAPto give the corresponding succinic
ester (12). Deprotection of the quinone with cerric ammonium ni-
trate afforded the target analogue 13.
1
The coupled products 14 and 15 were characterised by H,
13C, and two-dimensional NMR techniques, as well as by mass
spectrometry. The H NMR spectrum of 14 showed two amide
resonances (5.77 and 6.40 ppm) and a multiplet at 4.55 ppm corre-
sponding to the -proton of the lysine residue. Aseries of multiplets
at 1.35 (2H), 1.50 (2H), 1.69 (1H), 1.82 (1H), and 3.21 (2H) ppm
were also observed for the side chain methylenes of lysine.Analysis
1
1
of 15 by H NMR spectroscopy revealed three amide resonances
at 6.03, 6.74, and 7.08 ppm. The lysine  proton resonance was
observed as a multiplet at 4.53 ppm, while the lysine side chain
methylenes were observed as a series of multiplets at 1.34, 1.46,
1.71, 1.85, and 3.23 ppm. The  methylene of glycine was observed
as two doublet of doublets at 3.91 and 4.01 ppm.
Conclusions
A
new and efficient synthesis of 1-methyl-2,3,4,5-tetra-
methoxybenzene (2), and hence 1-bromo-2-methyl-3,4,5,6-
tetramethoxybenzene (1), from the commercially available
2-methyl-5,6-dimethoxy-1,4-benzoquinone (3) has been developed.
The reported procedure is quick and simple to carry out, the products
are obtained in high yield and they do not require purification by
chromatography. As such, this approach should be amenable to the
large scale synthesis of these important precursors to coenzyme Q
and analogues thereof. The synthesis of two side-chain functionalized
analogues of CoQ from 1 is also described. We report an improved
route to analogue 1022,24 and a preparation of the previously unknown
analogue 13. We have also have carried out studies on the coupling
of 10 to lysine derivatives as a first step in the development of an
immunoassay for the detection of coenzyme Q in biological tissues.
This is an important goal since an assay of this type would allow the
study and monitoring of diseases associated with coenzyme Q.
Experimental
General procedures
Scheme 3 i. AgNO3, NaOH, EtOH, 78%; ii. CAN, MeCN/H2O, 0 °C–
R.T., 55%; iii. NaBH4, MeOH, 0 °C–R.T., 74%; iv. succinic anhydride,
DMAP, Et3N, CH2Cl2, 37%; v. CAN, MeCN/H2O, 0 °C–R.T., 90%.
Proton detected NMR spectra were obtained on an Inova 500 spec-
trometer. Carbon detected NMR spectra were obtained on a Varian
XL300 spectrometer or a Varian Unity 300 spectrometer both oper-
ating at 75 MHz. Chemical shifts are reported in parts per million
(ppm), on the  scale, and are referenced to the appropriate solvent
peaks: CDCl3 referenced to (CH3)4Si at  0 ppm for 1H spectra, and
CDCl3 at  77.0 ppm for 13C spectra. All 13C NMR spectra were
obtained with a delay (D1) of 1 s. 1H and 13C NMR spectra were as-
signed by comparison, with all assignments verified by 2-D NMR
Model studies were next carried out to investigate the feasibil-
ity of coupling a coenzyme Q analogue of this type to a protein
as a first step to the development of an ELISA-based method for
the detection of natural coenzyme Q, Scheme 4. We chose to use
N-acetyl-L-lys methyl ester and a N-acetyl-gly-L-lys methyl ester
for these studies, where the lysine side chain allows coupling to the
2 3 7 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 3 7 1 – 2 3 7 5

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experiments. Mass spectrometry was performed by Electron Impact
(EI) at 4 kV using a Kratos MS80 Mass Spectrometer. All amino
acid conjugates were analysed by electrospray mass spectrometry
using a Micromass LCT spectrometer. IR spectra were obtained
using a Shimadzu 8201PC series FTIR, oils were analysed neat on
KBr disks. Unless otherwise stated all reactions were carried out
in oven-dried, or flame-dried glassware under an N2 atmosphere.
Radial chromatography was carried out on 1 mm, 2 mm or 4 mm
plates of Merck Silica Gel 60 PF254 containing gypsum, and visu-
alised with short-wavelength ultraviolet light. Dichloromethane
was distilled over calcium hydride, while diethyl ether and tetra-
hydrofuran were freshly distilled from sodium benzophenone ketal,
under a nitrogen atmosphere before use. Coenzyme Q-0 (3) was
obtained from Sigma Chemical Co®, and geranyl bromide was ob-
tained from Aldrich Chemical Co®. Butyllithium was used fresh or
titrated before use to determine exact concentration.
give the crude product. Purification by radial chromatography elut-
ing with 1:19 ethyl acetate:petroleum ether afforded 515,23 (426 mg,
48%) as a yellow oil; max/cm−1 2932, 2860, 1468; H(500 MHz;
CDCl3; Me4Si) 1.57 (3H, s, CCH3), 1.64 (3H, s, CCH3), 1.77
(3H, s, CCH3), 2.00 (2H, m, CH2), 2.07 (2H, m, CH2), 2.14 (3H,
s, ArMe), 3.32 (2H, d, J 6.8, ArCH2), 3.78 (3H, s, OMe), 3.79 (3H,
s, OMe), 3.90 (3H, s, OMe), 3.91 (3H, s, OMe), 5.04 (2H, m,
2 × CH); C(75 MHz; CDCl3; CDCl3) 11.61, 16.11, 17.61, 25.62,
25.73, 26.55, 39.64, 60.61, 61.01, 61.03, 107.01, 122.88, 124.23,
125.35, 129.23, 131.25, 134.93, 144.63, 144.87, 147.61, 147.80;
m/z (EI) 348.2306 (C21H32O4 requires 348.2301).
3-Bromo-2,6-dimethyl-8-(2-methyl-3,4,5,6-tetramethoxy-
phenyl)oct-6-en-2-ol (6). A solution of 5 (420 mg, 1.20 mmol)
in THF (40 cm3) was cooled in an ice bath and water was added
slowly to the reaction until it remained turbid. THF was then added
dropwise until the reaction just cleared. N-Bromosuccinimide
(236 mg, 1.33 mmol, 1.1 equiv) was added and the reaction stirred
at room temperature for 3 h. The reaction was extracted with ether
(×3), dried (MgSO4) and solvent removed to give the crude prod-
uct. Purification by radial chromatography, eluting with 1:9 ethyl
acetate:petroleum ether to 1:4 ethyl acetate:petroleum ether gave
the bromohydrin 6 (347 mg, 65%) as a yellow oil; max/cm−1 3477,
2936, 1740; H(500 MHz; CDCl3; Me4Si) 1.32 (3H, s, C(OH)CH3)
1.33 (3H, s, C(OH)CH3), 1.77 (3H, s, CCH3), 1.79 (1H, m,
CH2aCBr), 1.98 (1H, m, CH2bCBr), 2.09 (1H, m, CCH2a), 2.35
(1H, m, CCH2b), 2.14 (3H, s, ArMe), 3.33 (2H, m, ArCH2), 3.79
(3H, s, OMe), 3.78 (3H, s, OMe), 3.90 (3H, s, OMe), 3.91 (3H,
s, OMe), 3.95 (1H, dd, J 1.5 and 11.7, CHBr), 5.13 (1H, t, J 6.4,
CH); C(75 MHz; CDCl3; CDCl3) 11.78, 16.14, 25.81, 25.86,
26.62, 32.10, 38.23, 60.70 (×2), 61.10 (×2), 70.85, 72.44, 124.42,
125.28, 128.85, 133.24, 144.70, 145.00, 147.62, 147.87; m/z (EI)
444.1516 (C21H33O579Br requires 444.1509).
1-Bromo-2-methyl-3,4,5,6-tetramethoxybenzene (1). To a
stirred solution of 2 (490 mg, 2.30 mmol, 1 equiv) in dichloro-
methane (2.3 cm3) at 3–5 °C was added slowly a solution of bromine
(0.12 cm3, 2.30 mmol, 1 equiv) in dichloromethane (0.46 cm3). The
resulting dark orange solution was stirred for a further 5 min. The
reaction mixture was washed with water (3×), dilute aqueous
NaOH (3×), water, dried (MgSO4) and solvent removed to give 1
(566 mg, 84%) as a yellow oil; max/cm−1 2937, 2858; H(500 MHz;
CDCl3; Me4Si) 2.30 (3H, s, ArMe), 3.78 (3H, s, OMe), 3.85 (3H, s,
OMe), 3.91 (6H, s, 2 × OMe); C(75 MHz; CDCl3; CDCl3) 15.98,
60.86, 60.90, 61.15, 61.39, 114.32, 127.31, 145.50, 146.35, 147.23,
148.31; m/z (EI) 290.0163 (C11H15O479Br requires 290.0151).
1-Methyl-2,3,4,5-tetramethoxybenzene (2). 2,3-Dimethoxy-
5-methyl-1,4-benzoquinone 3 (1 g, 5.49 mmol, 1 equiv) was dis-
solved in hot ethanol and cooled rapidly in ice. To this was added
a solution of stannous chloride (4.95 g, 0.02 mol, 4 equiv) in conc.
HCl (5 cm3) during which time the red colour faded to pale yel-
low. This was extracted with ethyl acetate (3×), dried (MgSO4) and
solvent removed to give pale yellow crystals. This was suspended
in dimethylsulfate (7.2 cm3, 0.08 mmol, 14 equiv) and stirred in a
two-necked flask under nitrogen. To this was added portionwise a
solution of potassium hydroxide (12 g, 0.22 mol, 40 equiv) in water
(26 cm3) not allowing the solution to reflux. During this addition
a thick white precipitate formed, which then redissolved leaving
a yellow solution that faded to white. Upon completion of the ad-
dition of potassium hydroxide the reaction was heated to 80 °C
under a nitrogen atmosphere for 1 h, then allowed to cool slowly.
The reaction was diluted with water (180 cm3) and extracted with
ether (3×). The combined organic fractions were washed with 5%
aqueous NaOH, water, dried (MgSO4) and solvent removed to af-
ford 215 (990 mg, 85%) as a pale yellow oil; max/cm−1 2937, 2837,
1591; H(500 MHz; CDCl3; Me4Si)  2.22 (3H, s, ArMe), 3.78 (3H,
s, OMe), 3.81 (3H, s, OMe), 3.86 (3H, s, OMe), 3.93 (3H, s, OMe),
6.44 (1H, s, ArH); C(75 MHz; CDCl3; CDCl3) 15.84, 56.10, 60.65,
61.08, 61.17, 108.31, 125.89, 140.79, 145.40, 147.00, 149.11; m/z
(EI) 212.1057 (C11H16O4 requires 212.1049).
3-[3-Methyl-5-(2-methyl-3,4,5,6-tetramethoxyphenyl)pent-3-
enyl]-2,2-dimethyloxirane (7). To a stirred solution of 6 (100 mg,
0.22 mmol) in methanol (0.25 cm3) was added potassium carbonate
(2.17 g, 0.02 mol, 70 equiv) and the reaction was stirred at room
temperature for 16 h. The reaction was filtered and solvent re-
moved. Water was added and the resulting solution was extracted
with dichloromethane, dried (MgSO4) and solvent removed under
reduced pressure to give 722 (64 mg, 78%) as a pale yellow oil;

max/cm−1 2961, 2934, 2862, 2829; H(500 MHz; CDCl3; Me4Si)
1.24 (3H, s, CCH3), 1.25 (3H, s, CCH3), 1.64 (2H, m, CH2CO) 1.80
(3H, s, CCH3), 2.10 (2H, m, CCH2), 2.17 (3H, s, ArMe), 2.67
(1H, t, J 6.4, CHO), 3.32 (2H, d, J 2.9, ArCH2), 3.78 (3H, s, OMe),
3.79 (3H, s, OMe), 3.90 (3H, s, OMe), 3.91 (3H, s, OMe), 5.10 (1H,
t, J 5.4, CH); C(75 MHz; CDCl3; CDCl3) 11.62, 16.07, 18.59
(2×), 24.67, 25.67, 27.26, 36.19, 58.17, 60.51, 60.90, 60.93, 63.99,
123.37, 125.12, 128.79, 133.94, 144.57, 144.88, 147.50, 147.73;
m/z (EI) 364.2254 (C21H32O5 requires 364.2250).
4-Methyl-6-(2-methyl-3,4,5,6-tetramethoxyphenyl)hex-4-enal
(8). To a stirred solution of 7 (120 mg, 0.33 mmol) in diethyl ether
(16 cm3) was added a solution of periodic acid (75 mg, 0.33 mmol,
1 equiv) in THF (8 cm3). This was stirred at room temperature for
1.5 h during which time a fine precipitate formed. The reaction was
quenched via the addition of water, the ether layer was separated and
the aqueous phase extracted with ether (×2). The combined organic
fractions were dried (MgSO4) and solvent removed under reduced
pressure to afford 8 (90 mg, 85%) as a pale yellow oil (Found: C,
66.9; H, 7.9. Calc. for C18H26O5: C, 67.1; H, 8.1%); max/cm−1 2936,
1724; H(500 MHz; CDCl3; Me4Si) 1.79 (3H, s, CCH3), 2.12 (3H,
s, ArMe), 2.33 (2H, t, J 7.8, CH2CH2CHO), 2.52 (2H, dt, J 2.0 and
7.3, CH2CHO), 3.32 (2H, d, J 5.9, ArCH2), 3.78 (3H, s, OMe), 3.79
(3H, s, OMe), 3.90 (3H, s, OMe), 3.91 (3H, s, OMe), 5.09 (1H, t, J
6.4, CH), 9.74 (1H, t, J 1.9, CHO); C(75 MHz; CDCl3; CDCl3)
11.72, 16.27, 25.76, 31.79, 42.13, 60.68 (×2), 61.07 (×2), 123.99,
125.27, 128.62, 133.00, 144.68, 145.07, 147.62, 147.85, 202.47; m/z
(EI) 322.1771 (C18H26O5 requires 322.1780).
2,6-Dimethyl-8-(2-methyl-3,4,5,6-tetramethoxyphenyl)octa-
2,6-diene (5). A solution of 1 (735 mg, 2.50 mmol) in diethyl ether
(4.2 cm3) was cooled to 0 °C under an Ar atmosphere, and nBuLi
(1.6 M solution in hexanes, 1.8 cm3, 1.15 equiv) was added slowly.
During this addition the reaction went green then dark red and after
stirring at 0 °C for 30 min a pale precipitate formed. To this solu-
tion copper bromide dimethylsulfide complex (360 mg, 0.7 equiv)
was added, and stirring continued for 2.5 h during which time the
solution turned dark purple. To this geranyl bromide (0.55 cm3, 2.78
mmol, 1.1 equiv) in ether (2.8 cm3) was slowly added and the reac-
tion stirred at 0 °C for 2 h and room temperature for 3 h. The reaction
was quenched via the addition of 10% aqueous HCl and the ether
layer separated. The aqueous phase was extracted with ether (×3),
and the combined organic fractions were washed with saturated
aqueous NaHCO3, water, dried (MgSO4) and solvent removed to
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 3 7 1 – 2 3 7 5
2 3 7 3

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4-Methyl-6-(2-methyl-3,4,5,6-tetramethoxyphenyl)hex-4-
enoic acid (9). To a solution of silver nitrate (82 mg, 0.54 mmol,
1.5 equiv) in ethanol (3.9 cm3) stirring at room temperature under a
nitrogen atmosphere was added a solution of 8 (115 mg, 0.36 mmol)
in ethanol (2.3 cm3). To this was added dropwise a solution of NaOH
(0.18 cm3 of a 5 N aqueous solution) in ethanol (2.3 cm3) over a
period of 10 min. The resulting black solution was stirred at room
temperature for 16 h. The reaction was filtered and ethanol removed
under reduced pressure. Water was added, and the solution acidified
and extracted with ethyl acetate (2×). Combined organic fractions
were dried (MgSO4) and solvent removed to afford 924 (94 mg,
78%) as a yellow oil (Found: C, 64.1; H, 7.8. Calc. for C18H26O6: C
63.9; H 7.7%); max/cm−1 2936, 2660, 1709; H(500 MHz; CDCl3;
Me4Si) 1.79 (3H, s, CCH3), 2.12 (3H, s, ArMe), 2.31 (2H, t, J
7.3, CCH2), 2.45 (2H, t, J 6.8, CH2CO2H), 3.32 (2H, d, J 6.4,
ArCH2), 3.77 (3H, s, OMe), 3.78 (3H, s, OMe), 3.89 (3H, s, OMe),
3.90 (3H, s, OMe), 5.09 (1H, t, J 6.8, CH); C(75 MHz; CDCl3;
CDCl3) 11.69, 16.13, 25.75, 32.67, 34.27, 60.68 (×2), 61.08 (×2),
123.98, 125.32, 128.72, 133.03, 144.65, 145.02, 147.62, 147.83,
178.47; m/z (EI) 338.1729 (C18H26O6 requires 338.1729).
dried (MgSO4) and the solvent removed under reduced pressure.
Purification by radial chromatography eluting with 1:9 ethyl
acetate:petroleum ether afforded 12 (24 mg, 37%) as a pale yellow
oil (Found: C 62.4; H 7.7. Calc. for C22H32O8: C, 62.3; H, 7.6%);

max/cm−1 2937, 1738; H(500 MHz; CDCl3; Me4Si) 1.73 (2H, m,
CH2CH2O), 1.76 (3H, s, CCH3), 2.03 (2H, t, J 6.8, CCH2), 2.12
(3H, s, ArMe), 2.59 and 2.65 (4H, m, COCH2CH2CO2H), 3.30 (2H,
d, J 6.4, ArCH2), 3.78 (3H, s, OMe), 3.78 (3H, s, OMe), 3.89 (3H, s,
OMe), 3.90 (3H, s, OMe), 4.05 (2H, dt, J 2.4 and 6.8, CH2O), 5.05
(1H, t, J 6.4, CH); C(75 MHz; CDCl3; CDCl3) 11.66, 15.98, 25.71,
26.19, 26.69, 28.87, 35.65, 60.65, 61.05, 64.46, 64.66, 123.57,
125.32, 126.98, 128.92, 133.71, 144.59, 144.90, 147.48, 147.78,
172.22, 176.96; m/z (EI) 424.2129 (C22H32O8 requires 424.2097).
4-Methyl-6-(3-methyl-5,6-dimethoxy-1,4-benzoquinon-2-yl)-
hex-4-enyl hydrogen succinate (13). To a solution of 12 (24 mg,
0.06 mmol) in a 2:1 acetonitrile:water mixture (0.25 cm3) was
added a cooled solution of cerric ammonium nitrate (CAN) (77 mg,
0.14 mmol, 2.5 equiv) in a 1:1 acetonitrile:water mixture (0.3 cm3),
over a 10 min period. The resulting solution was stirred at 0 °C for
20 min and room temperature for 10 min before quenching via the
addition of water. The resulting reaction mixture was extracted with
dichloromethane (×2) and the combined organic fractions washed
with water, dried (MgSO4) and the solvent removed under reduced
pressure. Purification by radial chromatography eluting with di-
chloromethane to 1:19 MeOH:dichloromethane gave 13 (20 mg,
90%) as an orange oil; H(500 MHz; CDCl3; Me4Si) 1.72 (2H, m,
CH2CH2O), 1.74 (3H, s, CCH3), 2.02 (3H, s, ArMe), 2.03 (2H,
m, CCH2), 2.60–2.66 (4H, m, COCH2CH2CO2H), 3.18 (2H, d, J
6.8, ArCH2), 3.98 (3H, s, OMe), 4.00 (3H, s, OMe), 4.03 (2H, t, J
6.3, CH2O), 4.95 (1H, t, J 7.1, CH); C(75 MHz; CDCl3; CDCl3)
11.95, 16.12, 25.32, 26.58, 28.79, 28.82, 35.66, 61.14 (×2), 64.29,
119.73, 136.27, 138.94, 141.38, 144.21, 144.34, 172.13, 177.24,
183.93, 184.74; m/z (EI) 394.1621 (C20H26O8 requires 394.1623).
4-Methyl-6-(3-methyl-5,6-dimethoxy-1,4-benzoquinon-2-
yl)hex-4-enoic acid (10). To a solution of 9 (90 mg, 0.27 mmol) in a
2:1 acetonitrile:water mixture (1.1 cm3) was added a cooled solution
of cerric ammonium nitrate (CAN) (365 mg, 0.66 mmol, 2.5 equiv)
in a 1:1 acetonitrile:water mixture (1.3 cm3), over a 10 min period.
The resulting solution was stirred at 0 °C for 20 min and room
temperature for 10 min before quenching via the addition of water.
The resulting reaction mixture was extracted with dichloromethane
(×2) and the combined organic fractions washed with water, dried
(MgSO4) and the solvent removed under reduced pressure. Purifica-
tion by radial chromatography eluting with dichloromethane to 1:19
MeOH:dichloromethane gave 1024 (45 mg, 55%) as a bright orange
oil; H(500 MHz; CDCl3; Me4Si) 1.77 (3H, s, CCH3), 2.00 (3H, s,
ArMe), 2.30 (2H, m, CCH2), 2.43 (2H, m, CCH2CH2), 3.19 (2H,
d, J 6.8, ArCH2), 3.98 (3H, s, OMe), 3.99 (3H, s, OMe), 5.00 (1H, m,
CH); C(75 MHz; CDCl3; CDCl3) 11.88, 16.13, 25.17, 32.61, 34.19,
61.09 (×2), 120.02, 135.50, 139.01, 141.17, 144.17, 144.27, 179.03,
183.79, 184.60; m/z (EI) 308.1263 (C16H20O6 requires 308.1260).
Methyl
2-acetylamino-6-[4-methyl-6-(3-methyl-5,6-di-
methoxy-1,4-benzoquinon-2-yl)hex-4-enoylamino]hexanoate
(14). To a solution of 10 (10 mg, 0.032 mmol) and N--acetyl-L-
lys methyl ester hydrochloride (8.5 mg, 0.036 mmol, 1.1 equiv) in
dichloromethane (0.3 cm3) stirring under an Ar atmosphere, was
added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide·HCl (EDCI)
(8 mg, 0.042 mmol, 1.3 equiv), 1-hydroxybenzotriazole hydrate
(6.5 mg, 0.048 mmol, 1.5 equiv) and diisopropylethylamine (6 L,
0.035 mmol, 1.1 equiv). The resulting solution was stirred at room
temperature for 16 h. The reaction was quenched via the addition of
3 M aqueous NaCl solution and the layers separated. The aqueous
phase was extracted with dichloromethane (×2), and combined or-
ganic fractions were dried (MgSO4), and the solvent removed under
reduced pressure. Purification by radial chromatography eluting
with 1:19 MeOH:dichloromethane afforded 14 (10 mg, 63%) as an
orange oil; H(500 MHz; CDCl3; Me4Si) 1.35 (2H, m, NHCH2CH2),
1.50 (2H, m, NHCH2CH2CH2), 1.68 (1H, m, CH2aCHCO2Me),
1.76 (3H, s, CCH3), 1.82 (1H, m, CH2bCHCO2Me), 2.01 (3H, s,
ArMe), 2.04 (3H, s, COMe), 2.24 (2H, m, CCH2), 2.29 (2H, m,
CCH2CH2), 3.18 (2H, d, J 6.8,ArCH2), 3.21 (2H, m, NHCH2), 3.74
(3H, s, CO2Me), 3.99 (3H, s, OMe), 4.00 (3H, s, OMe), 4.55 (1H, m,
CHCO2Me), 4.99 (1H, t, J 7.3, CH), 5.77 (1H, brs, NH), 6.40 (1H,
d, J 6.8, NH); C(75 MHz; CDCl3; CDCl3) 11.95, 16.23, 22.25, 23.05,
25.33, 28.84, 31.79, 35.13, 35.27, 38.68, 51.82, 52.38, 61.13, 61.16,
119.91, 136.23, 139.06, 141.24, 144.20, 170.30, 172.85, 172.98,
182.30, 183.90; m/z (ES) 492.2465 (C25H36N2O8 requires 492.2472).
4-Methyl-6-(2-methyl-3,4,5,6-tetramethoxyphenyl)hex-4-
en-1-ol (11). To a stirred solution of sodium borohydride (10 mg,
0.27 mmol, 1 equiv) in methanol (0.55 cm3) at 0 °C was added a
solution of 8 (88 mg, 0.27 mmol) in methanol (1.1 cm3). The reac-
tion was stirred and allowed to warm to room temperature. After
20 min saturated aqueous NH4Cl was added to quench the reaction,
and methanol was removed under reduced pressure. The resulting
aqueous phase was extracted with dichloromethane (×2), and the
combined organic fractions washed with saturated aqueous NaCl,
water, dried (MgSO4) and solvent removed under reduced pressure
affording 11 (65 mg, 74%) as a yellow oil (Found: C, 66.7; H, 8.6.
Calc. for C18H28O5: C, 66.6; H, 8.7%); max/cm−1 3435, 2936, 1466,
1408; H(500 MHz; CDCl3; Me4Si) 1.65 (2H, m, CH2CH2OH), 1.76
(3H, s, CCH3), 2.04 (2H, t, J 6.8, CCH2), 2.11 (3H, s, ArMe),
3.30 (2H, d, J 6.8, ArCH2), 3.59 (2H, t, J 6.4, CH2OH), 3.76 (3H, s,
OMe), 3.77 (3H, s, OMe), 3.88 (3H, s, OMe), 3.89 (3H, s, OMe),
5.06 (1H, dt, J 1.5 and 6.8, CH); C(75 MHz; CDCl3; CDCl3)
11.88, 16.08, 25.90, 30.77, 36.26, 60.94, 61.99, 61.24 (×2), 62.76,
123.24, 125.22, 129.03, 134.82, 144.72, 145.03, 147.62, 147.92;
m/z (EI) 324.1937 (C18H28O5 requires 324.1937).
4-Methyl-6-(2-methyl-3,4,5,6-tetramethoxyphenyl)hex-4-
enyl hydrogen succinate (12). A solution of succinic anhydride
(34 mg, 0.34 mmol, 2.1 equiv), N,N-dimethylaminopyridine (1 mg,
0.01 mmol, 0.05 equiv) and triethylamine (0.03 cm3, 0.24 mmol,
1.5 equiv) in dichloromethane (0.6 cm3) was added slowly to a
solution of 11 (52 mg, 0.16 mmol) in dichloromethane (1.6 cm3).
This was stirred at room temperature under an Ar atmosphere for
16 h. The reaction was diluted with dichloromethane and washed
with 2 N aqueous HCl, water, saturated aqueous NaHCO3, water,
Methyl 2-(2-acetylamino-acetylamino)-6-[4-methyl-
6-(3-methyl-5,6-dimethoxy-1,4-benzoquinon-2-yl)hex-
4-enoylamino]hexanoate (15). To a solution of 10 (10 mg,
0.03 mmol) and N-acetyl-gly-L-lys methyl ester acetate (11 mg,
0.033 mmol, 1.1 equiv) in dichloromethane (0.3 cm3) stirring
under an Ar atmosphere, was added 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide·HCl (EDCI) (7.5 mg, 0.039 mmol, 1.3 equiv),
1-hydroxybenzotriazole hydrate (6 mg, 0.045 mmol, 1.5 equiv) and
2 3 7 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 3 7 1 – 2 3 7 5

View Article Online
3 F. L. Crane and P. Navas, Mol. Aspects Med., 1997, 18, s1.
diisopropylethylamine (6 L, 0.033 mmol, 1.1 equiv). The resulting
solution was stirred at room temperature for 16 h. The reaction was
quenched via the addition of 3 M aqueous NaCl solution and the
layers separated. The aqueous phase was extracted with dichloro-
methane (×2), and combined organic fractions were dried (MgSO4),
and the solvent removed under reduced pressure. Purification by
radial chromatography eluting with 1:19 MeOH:dichloromethane
afforded 15 (6 mg, 34%) as an orange oil; H(500 MHz; CDCl3;
Me4Si) 1.34 (2H, m, NHCH2CH2), 1.46 (2H, m, NHCH2CH2CH2),
1.71 (1H, m, CH2aCHCO2Me), 1.76 (3H, s, CCH3), 1.85 (1H,
m, CH2bCHCO2Me), 2.01 (3H, s, ArMe), 2.05 (3H, s, COMe),
2.28 (2H, m, CCH2), 3.15 (1H, m, NHCH2a), 3.17 (2H, d, J 7.3,
ArCH2), 3.23 (1H, m, NHCH2b), 3.74 (3H, s, CO2Me), 3.91 (1H,
dd, J 5.4 and 16.1, COCH2aNH), 3.99 (3H, s, OMe), 4.00 (3H, s,
OMe), 4.01 (1H, dd, J 5.4 and 16.1, COCH2bNH), 4.53 (1H, m,
CHCO2Me), 4.98 (1H, t, J 6.8, CH), 6.03 (1H, brs, NH), 6.74
(1H, brs, NH), 7.08 (1H, d, J 7.3, NH); C(75 MHz; CDCl3; CDCl3)
11.97, 16.28, 22.17, 22.89, 25.37, 28.86, 31.21, 35.04, 35.23, 38.55,
43.13, 52.01, 52.45, 61.15, 61.18, 119.85, 136.27, 139.10, 141.25,
143.01, 144.32, 169.08, 170.91, 172.51, 173.01, 183.96, 184.65;
m/z (ES) 549.2677 (C27H39O9N3 requires 549.2686).
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We would like to thank Mr Bruce Clark for assistance with mass
spectrometry, and the University of Canterbury for funding. We also
thank Dr Mike Lever (Canterbury Health Laboratories) for some
helpful preliminary discussions.
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