Total synthesis of [13C]2-, [13C]3-, and [13C]5-isotopomers of xanthohumol, the principal prenylflavonoid from hops

Article abstract of DOI:10.1002/jlcr.3571

Xanthohumol [(E)-6′-methoxy-3′-(3-methylbuten-2-yl)-2′,4′,4″-trihydroxychalcone], he principal prenylated flavonoid from hops, has a complex bioactivity profile, and ¹³C-labeled isotopomers of this compound are of potential use as molecular probes and as analytical standards to study metabolism and mode of action. 1,3-[¹³C]₂-Xanthohumol was prepared by an adaptation of the total synthesis of Khupse and Erhardt in 7 steps and 5.7% overall yield from phloroglucinol by a route incorporating a cascade Claisen-Cope rearrangement to install the 3′-prenyl moiety from a 5′-prenyl aryl ether and an aldol condensation between 1-[¹³C]-2′,4′-bis(benzyloxymethyloxy)-6′-methoxy-3′-(3-methylbuten-2-yl)acetophenone and 1′-[¹³C]-4-(methoxymethyloxy)benzaldehyde. The ¹³C-atom in the methyl ketone was derived from 1-[¹³C]-acetyl chloride while that in the aryl aldehyde was derived from [¹³C]-iodomethane. Tri- and penta-¹³C-labeled xanthohumols were similarly prepared by applying minor modifications to the route.

Full text of DOI:10.1002/jlcr.3571

Total Synthesis of [13C]2-, [13C]3-, and [13C]5-Isotopomers of
Xanthohumol, the Principal Prenylflavonoid From Hops
Duncan C. Ellinwood,a Mohamed F. El-Mansy,a,b Layhna S. Plagmann,a Jan F.
Stevens,c,d Claudia S. Maier,a Adrian F. Gombart,c,e and Paul R. Blakemorea,*
aDepartment of Chemistry, Oregon State University, Corvallis, OR 97331.
bNational Research Centre, Department of Organometallic and Organometalloid
Chemistry, El Behoose Street, P. O. Box 12622, Cairo, Dokki, Egypt.
cLinus Pauling Institute, Oregon State University, Corvallis, OR 97331.
dDepartment of Pharmaceutical Sciences, Oregon State University, Corvallis, OR
97331.
eDepartment of Biochemistry and Biophysics, Oregon State University, Corvallis, OR
97331.
*Correspondence to: Paul R. Blakemore, Department of Chemistry, Oregon State
University, Corvallis, OR 97331, USA.
E-mail: paul.blakemore@oregonstate.edu
Abstract
Xanthohumol [(E)-6´-methoxy-3´-(3-methylbuten-2-yl)-2´,4´,4´´-trihydroxychalcone],
the principal prenylated flavonoid from hops, has a complex bioactivity profile and
13C-labelled isotopomers of this compound are of potential use as molecular probes
and as analytical standards to study metabolism and mode-of-action. 1,3-[13C]2-
Xanthohumol was prepared by an adaptation of the total synthesis of Khupse and
Erhardt in 7 steps and 5.7% overall yield from phloroglucinol by a route incorporating
a cascade Claisen-Cope rearrangement to install the 3´-prenyl moiety from a 5´-
prenyl aryl ether and an aldol condensation between 1-[13C]-2´,4´-
bis(benzyloxymethyloxy)-6´-methoxy-3´-(3-methylbuten-2-yl)acetophenone and 1´-
[13C]-4-(methoxymethyloxy)benzaldehyde. The 13C-atom in the methyl ketone was
derived from 1-[13C]-acetyl chloride while that in the aryl aldehyde was derived from
[13C]-iodomethane. Tri- and penta-13C-labelled xanthohumols were similarly prepared
by applying minor modifications to the route.
Keywords
phytochemicals, chalcones, aldol condensation, Claisen rearrangement, Cope
rearrangement
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Introduction
Xanthohumol (1) is the principal prenylated flavonoid found in the inflorescenes
('hops') of the female hop plant, Humulus lupulus L. (Figure 1).1 Because hops are
utilized during the manufacture of beer to add a characteristic floral bitterness,
xanthohumol and compounds derived from it during the brewing process find their
way into the human diet and they have been implicated in a variety of health effects,
both beneficial and nefarious. For example, xanthohumol itself has received attention
as a cancer chemopreventative agent2 with indications against metabolic syndrome,3
while the cyclized form of desmethylxanthohumol, 8-prenylnaringenin, is the most
potent phytoestrogen known.4 The biochemistry of xanthohumol is nuanced and
there is a need for probe molecules to help dissect the role that xanthohumol
metabolism has on its biological mode-of-action. Fortunately, the simple chalcone
structure of xanthohumol makes the prospect of preparing analogs for SAR and
other experiments enticing. Of note, although it is commonly depicted as a
keto/phenol tautomer (1), the intense yellow color of xanthohumol (in the solid state
and in solution) and the presence of a far downfield signal in its 1H NMR spectrum
(H = 14.7 ppm), suggests that the true nature of this compound is the fully
conjugated enol/orthoquinomethide tautomer 1´.
In connection with ongoing studies of its biological activity,3b-e we required 13C-
labelled isotopomers of xanthohumol as standards for mass spectrometric isotope
dilution analyses and as probe molecules to track the fate of xanthohumol and its
downstream metabolites in a mouse model for metabolic syndrome. Surprisingly,
although a synthesis of a dimethylated congener of xanthohumol was reported quite
some time ago,5 a total synthesis of this important chalcone, an effort by Khupse and
,
Erhardt, did not appear until 2007.6 7 The Khupse-Erhardt elaboration employs a late-
stage aldol condensation between a methyl ketone fragment 2 (PG = MOM) and an
aryl aldehyde fragment 3 (PG´ = MOM) to generate a protected form of the chalcone
natural product 1. Given the practicality of the Khupse-Erhardt xanthohumol
synthesis, and the fact that a modified version of it was recently successfully used by
Fang and coworkers to access xanthohumol analogs with improved anticancer
activity,8 we elected to adapt it for the synthesis of a series of 13C-labelled
xanthohumol isotopomers. Herein, we report execution of this plan and detail
syntheses of [13C]2-, [13C]3-, and [13C]5-xanthohumols employing conveniently
obtained isotopically enriched starting materials.9
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Results and Discussion
We desired access to xanthohumol isotopomers with at least two 13C-atoms
distributed within different regions of the molecule such that metabolic bond
cleavage reactions could be easily detected. The first target of interest was 1,3-
[13C]2-xanthohumol ([13C]2-1) and its synthesis called for suitably protected methyl
aryl ketone 2 and aryl aldehyde 3 fragments with 13C labels at their respective
carbonyl group carbon-atoms. Our initial plan was to replace the methoxymethyl
(MOM) protecting groups used in the Khupse-Erhardt synthesis wholly with
benzyloxymethyl (BOM) protecting groups to avoid use of the more noxious
chloromethyl ether reagent require to install MOM versus BOM groups. BOM
protecting groups served well for the requisite methyl aryl ketone compound ([13C]-
11) but substitution of MOM for BOM in the aryl aldehyde fragment synthesis was
ultimately precluded when it was identified that the optimal route to this material
necessitated a benzylic oxidation step incompatible with a BOM group.
Synthesis of the methyl aryl ketone fragment began with Friedel-Crafts acetylation of
phloroglucinol (1,3,5-trihydroxybenzene, 4) with 1-[13C]-acetyl chloride (Scheme 1).10
As anticipated based on the comparable known reaction with MOMCl,6 exposure of
the so-generated labelled phloracetophenone [13C]-5 with an excess of BOMCl
resulted in alkylation of only two out of the three phenolic hydroxyl groups to yield
bis-BOM ether [13C]-6. The recalcitrance of the remaining phenol to react is likely
due to engagement of the hydroxyl group in a hydrogen-bond with the adjacent
carbonyl group. As a prelude to a Claisen-Cope rearrangement cascade to
regiospecifically install the C3´ prenyl group,11 phenol [13C]-6 was next converted into
its prenyl ether derivative [13C]-8. As previously observed by Khupse and Erhardt,6
we found that this operation was best achieved using Mitsunobu conditions (as
illustrated) while a traditional Williamson ether synthesis (prenyl bromide, K2CO3,
acetone, reflux) gave less than 50% conversion to the desired ether even after
extended reaction times.
With aryl allyl ether [13C]-8 in hand, double transposition of the prenyl group from O6´
to C3´ via a Claisen-Cope rearrangement was investigated. Khupse and Erhardt
reported that this task could be achieved from the analogous bis-MOM protected
compound in 64% yield by direct heating in N,N-dimethylaniline;6,12 however, Fang et
al. reported only a 31% yield for the exact same transformation.8 In the case of [13C]-
8, we also encountered problems with the simple thermal process and obtained at
best a 25% yield (PhNMe2, 200 °C, 2 h) of the desired rearrangement product [13C]-9
which was accompanied by unreacted starting material, the intermediate C5´
isoprenylated arene, and innumerable minor side-products. Longer reaction times
only led to decomposition and even lower isolated yields of prenylated arene [13C]-9.
A better outcome (47% yield, 0.15 mmol scale) was obtained using microwave
irradiation in 1,2-dichlorobenzene (180 °C, 1.5 h) but the reaction was not
reproducible on a practically relevant scale (≥1 mmol). After further experimentation,
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the Eu(III) catalyzed rearrangement conditions of Metz et al.13 were found to
consistently afford [13C]-9 in ca. 40% isolated yield even on multigram scale.
Unfortunately, [13C]-9 was the minor product formed using Eu(fod)3 (Resolve-AlTM)
catalysis and the bulk of the mass balance was dihydrobenzopyran derivative [13C]-
10, an apparent result of oxymetalation/protodemetalation from the putative
intermediate C5´ isoprenylated arene. The spontaneous formation of
dihydrobenzofurans such as [13C]-10 following Claisen rearrangement of O-prenyl
arenes has been observed previously under a variety of different reaction
conditions.14 Resubjection of dihydrobenzopyran [13C]-10 to the reaction conditions
did not result in its further conversion to [13C]-9. Methylation of [13C]-9 was uneventful
and gave a good yield of the requisite methyl ketone fragment [13C]-11 under
standard conditions.
Prenylated chalcones have been been successfully accessed by Suzuki-Miyaura
cross-coupling of appropriate aryl iodides with prenyl boronic esters.15 Accordingly,
we briefly explored an alternate preparation of [13C]-11 via iodination of [13C]-6
followed by cross-coupling etc. This plan was thwarted with the discovery that
iodination of [13C]-6 (N-iodosuccinimide, DMF, rt, 4.5 h, 78% yield) occurred
exclusively at the undesired C5´ position while iodination of the O-methylated
derivative of [13C]-6 gave a 62:38 mixture of regioisomeric aryl iodides, favoring
again the undesired C5´ halogenated adduct.
Our preferred strategy to a suitable labelled aryl aldehyde fragment, [13C]-16,
involved benzylic oxidation of MOM protected p-cresol [13C]-14 such that [13C]-methyl
iodide could provide a convenient and inexpensive source of the 13C-atom (Scheme
2). Accordingly, [13C]-14 was prepared from bromobenzene 13 by bromine-lithium
exchange followed by alkylation of the intermediate aryllithium with [13C]-methyl
iodide. This reaction also generated minor quantities of the reduced bromide
(MOMOPh, 11%) and the butylated arene (4-MOMOC6H4Bu, 13%) resulting from
attack of the aryllithium upon the bromobutane by-product of halogen-metal
exchange. Direct conversion of cresol derivative [13C]-14 to the target aldehyde [13C]-
16 was attempted both via an IBX mediated oxidation (≤10% yield [13C]-16)16 and via
a TEMPO/Co(OAc)2 catalyzed oxidation (no reaction),17 but neither method gave an
acceptable result. Instead, [13C]-14 was successfully converted to aldehyde [13C]-16
over two steps by employing a Wohl-Ziegler radical bromination18 followed by a
Kornblum oxidation.19 The intermediate bromide [13C]-15 (which was co-generated
with variable quantities of the corresponding dibromide 3-25%) was found to
decompose significantly within 12 hours and so it was treated immediately upon
isolation with warm dimethylsulfoxide to transform it into the desired aldehyde [13C]-
16. Being quite susceptible to aerial oxidation, the aldehyde was likewise regarded
as a labile intermediate and so it was also used as quickly as possible in the next
step. Indeed, it was found to be optimal for bromination, Kornblum oxidation, and
aldol condensation to be performed during the same day if at all possible.
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Completion of the total synthesis of 1,3-[13C]2-xanthohumol called for aldol
condensation between the previously assembled aryl methyl ketone and aryl
aldehyde fragments followed by removal of the collection of BOM and MOM
protecting groups (Scheme 3). Minor modifications to the original Khupse-Erhardt
procedures were required to achieve this end game in an acceptable overall yield.
For example, possibly due to the heightened lipophilicity of bis-BOM group protected
ketone [13C]-11, aldol condensation between this compound and aryl aldehyde [13C]-
16 in basic aqueous methanol required considerably longer (16 h) to achieve than
the 4 h reported for the analogous reaction involving bis-MOM group protected
ketone 2 (PG = MOM).6 In the case of the final deprotection step, we found that the
conditions used by Khupse and Erhardt to remove three MOM groups (c. HCl, aq.
MeOH, rt) did not work well (≤20% yield) for the hydrolysis of two BOM groups and
one MOM group from aldol reaction product [13C]2-17. After extensive
experimentation, it was discovered that conditions reported by Hall and
Deslongchamps for MOM group hydrolysis (c. HCl, i-PrOH, 60 °C) gave acceptable
results and provided a 45-60% yield of the desired labelled xanthohumol [13C]2-1;20
however, small quantities of HCl addition adduct [13C]2-18 were co-generated
alongside the desired target. Alkene [13C]2-1 and chloroalkane [13C]2-18 possess
very similar polarity and they are difficult to separate from one another. Nonetheless,
a two-stage column chromatography protocol was developed that reliably delivers
[13C]2-1 in a completely pure form free of [13C]2-18 and any other contaminants from
the aforegoing chemistry (see experimental section for details).
Aside from the expected spectral differences due to 13C incorporation, the 1,3-[13C]2-
xanthohumol ([13C]2-1) generated from the route described above was otherwise
identical to a sample of commercially available natural xanthohumol. The presence
of the two sites of 13C-atom enrichment were clearly evident in mass (i.e., m/z
(M+H)+ = 357 for [13C]2-1), IR, and NMR spectra. In the IR spectrum, the carbonyl
stretching band was 17 cm–1 lower for [13C]2-1 as compared to natural 1. The 13C
NMR spectrum for [13C]2-1 (in d6-DMSO) showed strongly enriched signals for C1 (
= 191.7 ppm) and C3 ( = 142.5 ppm) while scalar coupling to 13C atoms was
observed for signals attributable to H2, H3, and H2´´ in the 1H NMR spectrum (Figure
2). Complete spectra data can be found below in the experimental section and full
NMR spectra are available in the Supporting Information.
Finally, the other desired more highly labelled isotopomers of xanthohumol were
prepared in an essentially identical fashion to [13C]2-1 by using additional 13C-
enriched starting materials at appropriate junctures in the total synthesis (Figure 3).
Thus, triple 13C-labelled xanthohumol ([13C]3-1) was synthesized by using [13C]-
iodomethane in the phenol methylation step (cf. [13C]2-9 to [13C]2-11, Scheme 1). To
access quintuple 13C-labelled xanthohumol ([13C]5-1), [13C]-iodomethane was again
incorporated into the phenol methylation step but now also 1,2-[13C]2-3-methyl-2-
buten-1-ol (prepared by the method of Lugtenburg)21 was used instead of unlabelled
prenyl alcohol (7) in the Mitsunobu reaction (cf. [13C]2-6 to [13C]2-8, Scheme 1). As for
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the double labelled isotopomer [13C]2-1, the two newly prepared compounds, [13C]3-1
and [13C]5-1, exhibited the expected characteristic spectral differences to natural
xanthohumol but they were otherwise identical.
Experimental
All commercially available reagents were used as received unless otherwise noted.
Preparative chromatographic separations were performed on silica gel 60 (35-75
m) and reactions followed by TLC analysis using silica gel 60 plates (2-25 m) with
fluorescent indicator (254 nm) and visualized with UV or phosphomolybdic acid.
Infra-red (IR) spectra were recorded in Fourier transform mode using an ATR probe
for solids, while oils were supported between NaCl plates (neat). 1H and 13C NMR
spectra were recorded in Fourier transform mode at the field strength specified and
from the indicated deuterated solvents in standard 5 mm diameter tubes. Chemical
shift in ppm is quoted relative to residual solvent signals calibrated as follows: CDCl3
H (CHCl3) = 7.26 ppm, C = 77.2 ppm; (CD3)2SO H (CD3SOCHD2) = 2.50 ppm, C =
39.5 ppm. Multiplicities in the 1H NMR spectra are described as: s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, br = broad. Numbers in parentheses
following carbon atom chemical shifts refer to the number of attached hydrogen
atoms determined by either DEPT or HSQC NMR experiments. Low (MS) and high
resolution (HRMS) mass spectra were obtained using either electon impact (EI) or
electrospray (ES) ionization techniques. Ion mass/charge (m/z) ratios are reported
as values in atomic mass units.
1-[13C]-2´,4´,6´-Trihydroxyacetophenone ([13C]-5). By a modification to the method
of Maier et al.10 To a stirred suspension of phloroglucinol (1.58 g, 12.5 mmol) in
CH2Cl2-nitromethane (1:1, 30 mL) at rt under Ar was added powdered AlCl3 (6.67 g,
50.0 mmol) in four portions (a slight exotherm was noted). The resulting
homogenous yellow/brown solution was stirred for 30 min and then treated with neat
1-[13C]-acetyl chloride (1.00 g, 12.6 mmol) in one portion. The resulting mixture was
heated to a gentle reflux and stirred for 3 h. After this time, the mixture was cooled to
rt and poured into ice water (100 mL). A majority of the low boiling volatile solvents
were removed using a rotatory evaporator and the residue was extracted with EtOAc
(3x20 mL). The combined organic extracts were washed with brine (20 mL), dried
(Na2SO4) and concentrated in vacuo. The residue was purified by column
chromatography (SiO2, eluting with 60% EtOAc in hexanes) to afford the desired
acylated product [13C]-5 (1.34 g, 7.93 mmol, 63%) as a tan powder: mp 207-209 °C;
IR (ATR) 3522, 3451, 3107, 1622, 1515, 1463, 1360, 1266, 1239, 1164, 1064 cm–1;
1H NMR (400 MHz, d6-DMSO)  12.23 (2H, s), 10.36 (1H, s), 5.80 (2H, s), 2.54 (3H,
d, 2JCH = 6.1 Hz) ppm; 13C NMR (100 MHz, d6-DMSO)  202.5 (0, enriched), 164.8
(0), 164.3 (2C, 0), 104.0 (0, d, 1JCC = 56 Hz), 94.5 (2C, 1), 32.4 (3, d, 1JCC = 41 Hz)
ppm; MS (EI+) m/z 169 (100%, [M]+•), 155 (82), 126 (35), 69 (70); HRMS (ES+) m/z
13
170.0536 (calcd. for 12C7 CH9O4: 170.0534).
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1-[13C]-2´,4´-Bis(benzyloxymethyloxy)-6´-hydroxyacetophenone ([13C]-6). A
stirred suspension of triol [13C]-5 (1.25 g, 7.40 mmol) in anhydrous CH2Cl2 (25 mL) at
0 °C under Ar was treated with diisopropylethylamine (7.56 mL, d = 0.76, 5.75 g,
44.5 mmol, 6 eq) followed by neat benzyl chloromethyl ether (BOMCl, 3.06 mL, d =
1.14, 3.49 g, 22.3 mmol, 3 eq) which was added dropwise during 4 min. The
resulting homogenous brown solution was allowed to warm to rt and stirred for 2.75
h. After this time, sat. aq. NaHCO3 (20 mL) was added and the mixture partitioned
between EtOAc (40 mL) and H2O (30 mL). The aqueous phase was extracted with
EtOAc (2x20 mL) and the combined organic phases washed successively with H2O
(30 mL) and brine (20 mL, then dried (Na2SO4) and concentrated in vacuo. The
residue was purified by column chromatography (SiO2, eluting with 20% EtOAc in
hexanes) to afford the desired bis-BOM ether [13C]-6 (2.48 g, 6.06 mmol, 82%) as
colorless oil: IR (neat) 3032, 2909, 1619, 1584, 1252, 1213, 1173, 1068, 830, 698
cm–1; 1H NMR (400 MHz, CDCl3)  13.74 (1H, s), 7.36-7.30 (10H, m), 6.38 (1H, dm,
J = 2.3 Hz), 6.34 (1H, dm, J = 2.3 Hz), 5.37 (2H, s), 5.29 (2H, s), 4.75 (2H, s), 4.71
(2H, s), 2.64 (3H, d, 2JCH = 6.1 Hz) ppm; 13C NMR (100 MHz, CDCl3)  203.6 (0,
enriched), 167.0 (0), 163.7 (0), 160.6 (0), 137.0 (0), 136.9 (0), 128.73 (2C, 1), 128.65
(2C, 1), 128.3 (1), 128.2 (3C, 1), 128.1 (2C, 1), 107.2 (0, d, 1JCC = 56 Hz), 97.5 (1),
94.4 (1), 92.5 (2), 92.0 (2), 71.1 (2), 70.6 (2), 33.2 (3, d, 1JCC = 42 Hz) ppm; MS (EI+)
m/z 409 (15%, [M]+•), 379 (17), 349 (8), 306 (13), 258 (12), 215 (10), 181 (18), 120
(22), 107 (32), 91 (100); HRMS (ES+) m/z 410.1705 (calcd. for 12C2313CH25O6:
410.1685).
1-[13C]-2´,4´-Bis(benzyloxymethyloxy)-6´-[(3-methylbuten-2-
yl)oxy]acetophenone ([13C]-8). A stirred solution of phenol [13C]-6 (3.61 g, 8.83
mmol) and triphenylphosphine (3.47 g, 13.2 mmol) in anhydrous THF (60 mL) at 0
°C under Ar, was treated with prenyl alcohol (3-methyl-2-buten-1-ol, 7, 1.15 mL, d =
0.86, 989 mg, 11.5 mmol) followed by the dropwise addition of diethyl
azodicarboxylate (DEAD, 5.23 mL, d = 0.956, 5.00 g, 40 wt.% in PhMe, 11.5 mmol)
during 3 min. The resulting mixture was allowed to warm to rt and stirred for 22 h.
After this time, the mixture was concentrated in vacuo and the residue was purified
by column chromatography (SiO2, eluting with 18-20% EtOAc in hexanes) to afford
the desired aryl prenyl ether [13C]-8 (3.32 g, 6.96 mmol, 79%) as a colorless oil: IR
(neat) 2912, 1663, 1605, 1454, 1168, 1057, 822, 738 cm–1; 1H NMR (400 MHz,
CDCl3)  7.34-7.28 (10H, m), 6.59-6.58 (1H, m), 6.37-6.36 (1H, m), 5.41 (1H, t of
septet, J = 6.6, 1.4 Hz), 5.26 (2H, s), 5.24 (2H, s), 4.71 (2H, s), 4.70 (2H, s), 4.49
(2H, d, J = 6.6 Hz), 2.48 (3H, d, 2JCH = 6.3 Hz), 1.75 (3H, s), 1.70 (3H, s) ppm; 13C
NMR (100 MHz, CDCl3)  202.1 (0, enriched), 159.8 (0), 157.4 (0), 155.5 (0), 138.1
(0), 137.26 (0), 137.22 (0), 128.62 (2C, 1), 128.58 (2C, 1), 128.2 (4C, 1), 128.1 (1),
128.0 (1), 119.5 (1), 116.5 (0, d, 1JCC = 54 Hz), 96.4 (1), 95.4 (1), 92.8 (2), 92.5 (2),
70.4 (2), 70.2 (2), 65.8 (2), 32.7 (3, d, 1JCC = 43 Hz), 25.9 (3), 18.4 (3) ppm; MS (EI+)
m/z 477 (4%, [M]+•), 408 (91), 378 (91), 348 (43), 305 (95), 257 (77), 214 (60), 181
(90), 91 (100); HRMS (ES+) m/z 478.2356 (calcd. for 12C2813CH33O6: 478.2311).
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1-[13C]-2´,4´-Bis(benzyloxymethyloxy)-6´-hydroxy-3´-(3-methylbuten-2-
yl)acetophenone ([13C]-9). A stirred solution of aryl prenyl ether [13C]-8 (2.64 g, 5.53
mmol) in chlorobenzene (50 mL) was treated with solid NaHCO3 (511 mg, 6.08
mmol) followed by europium(III) tris(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-
octanedionate) [Eu(fod)3, 574 mg, 0.554 mmol, 10 mol%). The resulting mixture was
heated to a gentle reflux and stirred for 3 h. After this time, the mixture was allowed
to cool to rt, concentrated in vacuo, and allowed to stand at rt for 18 h. The residue
was then purified by column chromatography (SiO2, eluting with 15-20% EtOAc in
hexanes) to afford, in order of elution, the desired prenylated acetophenone [13C]-9
(1.05 g, 2.20 mmol, ≤ 40%, ca. 90-95% pure) and the unwanted isomeric benzofuran
derivative [13C]-10 (1.07 g, 2.24 mmol, 41%), both as colorless oils.
Data for [13C]-9: IR (neat) 2912, 1612, 1577, 1362, 1253, 1043, 952, 737, 697 cm–1;
1H NMR (400 MHz, CDCl3)  12.99 (1H, s), 7.38-7.27 (10H, m), 6.57 (1H, s), 5.33
(2H, s), 5.13 (1H, tm, J = 5.9 Hz), 5.10 (2H, s), 4.70 (4H, s), 3.32 (2H, d, J = 6.4 Hz),
2.73 (3H, d, 2JCH = 6.1 Hz), 1.75 (3H, s), 1.67 (3H, s) ppm; 13C NMR (100 MHz,
CDCl3)  204.2 (0, enriched), 163.7 (0), 161.9 (0), 157.3 (0), 137.08 (0), 137.02 (0),
131.9 (0), 128.7 (2C, 1), 128.7 (2C, 1), 128.20 (1), 128.16 (1), 128.1 (2C, 1), 127.9
(2C, 1), 123.3 (1), 116.4 (0), 111.2 (0, d, 1JCC = 54 Hz), 99.4 (2), 99.2 (1), 91.9 (2),
72.7 (2), 70.6 (2), 31.8 (3, d, 1JCC = 42 Hz), 25.9 (3), 23.4 (2), 18.2 (3) ppm; MS (EI+)
m/z 477 (10%, [M]+•), 476 (55), 355 (83), 325 (98), 282 (98), 271 (50), 181 (31), 91
(100); HRMS (ES+) m/z 500.2160 (calcd. for 12C2813CH32O6Na: 500.2130).
Data for [13C]-10: IR (neat) 2965, 2909, 1607, 1455, 1409, 1353, 1190, 1061, 964,
738, 698 cm–1; 1H NMR (400 MHz, CDCl3)  7.37-7.26 (10H, m), 6.61 (1H, s), 5.32-
5.28 (2H, AB quartet), 5.27 (2H, s), 4.74 (2H, s), 4.72 (2H, s), 4.44 (1H, q, J = 6.6
Hz), 2.55 (3H, d, 2JCH = 6.2 Hz), 1.42 (3H, s), 1.38 (3H, d, J = 6.6 Hz), 1.18 (3H, s)
ppm; 13C NMR (100 MHz, CDCl3)  198.4 (0, enriched), 159.3 (0), 157.2 (0), 156.2
(0), 137.3 (0), 137.1 (0), 128.7 (2C, 1), 128.6 (2C, 1), 128.13 (2C, 1), 128.11 (1),
128.08 (2C, 1), 128.0 (1), 118.4 (0), 110.0 (0), 95.1 (1), 93.6 (2), 91.7 (2), 90.2 (1),
70.44 (2), 70.41 (2), 43.7 (0), 32.7 (3, d, 1JCC = 42 Hz), 25.7 (3), 21.1 (3), 14.4 (3)
ppm; MS (ES+) m/z 500 (M+Na)+; HRMS (ES+) m/z 500.2080 (calcd. for
12
C
28
13CH32O6Na: 500.2130).
1-[13C]-2´,4´-Bis(benzyloxymethyloxy)-6´-methoxy-3´-(3-methylbuten-2-
yl)acetophenone ([13C]-11). A stirred suspension of phenol [13C]-9 (705 mg, 1.48
mmol) and K2CO3 (2.04 g, 14.8 mmol, 10 eq) in acetone (15 mL) at rt was treated
with neat iodomethane (0.47 mL, d = 2.24, 1.05 g, 7.41 mmol, 5 eq). The resulting
mixture was stirred vigorously for 18 h and then concentrated in vacuo. The residue
was partitioned between EtOAc (20 mL) and H2O (15 mL) and the aqueous phase
extracted with EtOAc (10 mL). The combined organic phases were washed
successively with sat. aq. Na2S2O3 (10 mL), H2O (10 mL), and brine (10 mL), and
then dried (Na2SO4) and concentrated in vacuo. The residue was purified by column
chromatography (SiO2, eluting with 20% EtOAc in hexanes) to afford the desired
methyl aryl ether [13C]-11 (610 mg, 1.24 mmol, 84%) as a colorless oil: IR (neat)
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2917, 1660, 1599, 1454, 1163, 1045, 936, 737, 698 cm–1; 1H NMR (400 MHz, CDCl3)
 7.36-7.29 (10H, m), 6.64 (1H, s), 5.32 (2H, s), 5.17 (1H, tm, J = 6.8 Hz), 5.05 (2H,
s), 4.74 (2H, s), 4.71 (2H, s), 3.77 (3H, s), 3.34 (2H, d, J = 6.8 Hz), 2.48 (3H, d, 2JCH
= 6.2 Hz), 1.74 (3H, s), 1.66 (3H, s) ppm; 13C NMR (100 MHz, CDCl3)  202.3 (0,
enriched), 157.8 (0), 156.0 (0), 153.8 (0), 137.7 (0), 137.2 (0), 131.5 (0), 128.7 (2C,
1), 128.6 (2C, 1), 128.3 (2C, 1), 128.2 (1), 128.0 (2C, 1), 127.9 (1), 123.4 (1), 120.3
(0, d, 1JCC = 54 Hz), 117.5 (0), 99.3 (2), 95.2 (1), 92.4 (2), 71.7 (2), 70.2 (2), 56.1 (3),
32.9 (3, d, 1JCC = 42 Hz), 25.9 (3), 23.3 (2), 18.1 (3) ppm; MS (EI+) m/z 491 (3%,
[M]+•), 477 (18), 383 (30), 326 (26), 250 (23), 91 (100); HRMS (ES+) m/z 492.2487
(calcd. for 12C2913CH35O6: 492.2467).
1-Bromo-4-(methoxymethyloxy)benzene (13). A stirred solution of 4-bromophenol
(12, 12.2 g, 70.5 mmol) in anhydrous CH2Cl2 (300 mL) at 0 °C under Ar was treated
with diisopropylethylamine (27.0 mL, d = 0.76, 20.5 g, 158.9 mmol). Neat
chloromethyl methyl ether (MOMCl, 7.50 mL, d = 1.06, 7.08 g, 75.9 mmol, CARE!)
was then added dropwise during 10 min. The resulting dark orange solution was
allowed to stir for 2 h while warming to rt. After this time, any active alkylating agent
was quenched by the addition of conc. aq. NH3 (10 mL) and the reaction mixture
stirred for a further 10 min. The mixture was partitioned between EtOAc (80 mL) and
H2O (80 mL) and the aqueous phase extracted with EtOAc (2x60 mL). Combined
organic phases were then washed successively with H2O (40 mL) and brine (40 mL),
dried (Na2SO4), and concentrated in vacuo. The residue was purified by column
chromatography (SiO2, eluting with 15% EtOAc in hexanes) to afford the title MOM
ether 13 (12.65 g, 58.3 mmol, 83%) as a colorless oil: IR (neat) 2956, 2903, 1591,
1488, 1235, 998.8, 591, 508 cm–1; 1H NMR (400 MHz, CDCl3)  7.38 (2H, d, J = 9.0
Hz), 6.92 (2H, d, J = 9.0 Hz), 5.15 (2H, s), 3.47 (3H, 3) ppm; 13C NMR (100 MHz,
CDCl3)  156.5 (0), 132.5 (2C, 1), 118.2 (2C, 1), 114.4 (0), 94.7 (2), 56.2 (3) ppm. 1H
and 13C NMR spectral data are in agreement with those previously reported.22
4-(Methoxymethyloxy)-1-[13C]methylbenzene ([13C]-14). A stirred solution of aryl
bromide 13 (5.20 g, 24.0 mmol) in anhydrous THF (130 mL) at –78 °C was treated
dropwise with n-BuLi (15.8 mL, 2.10 M in hexanes, 33.2 mmol) during 10 min. The
resulting aryllithium was stirred for 10 min and then treated with neat [13C]-methyl
iodide (1.60 mL, d = 2.28, 3.65 g, 25.5 mmol). The reaction mixture was allowed to
stir while warming to rt for 2 h and then sat. aq. NH4Cl (15 mL) was added. The
quenched mixture was then partitioned between EtOAc (50 mL) and H2O (50 mL)
and the aqueous phase extracted with EtOAc (2x30 mL). The combined organic
phases were washed successively with conc. aq. NH3 (10 mL), brine (20 mL), and
then dried (Na2SO4) and concentrated in vacuo. The residue was purified by column
chromatography (SiO2, eluting with 10% EtOAc in hexanes) to afford an inseparable
mixture (3.15 g) of the methylated benzene [13C]-14 (69 wt.%, effectively 2.17 g, 14.2
mmol, 59%), 4-MOMOC6H4Bu (19 wt.%, eff. 599 mg, 3.08 mmol, 13%), and
MOMOPh (12 wt.%, eff. 378 mg, 2.74 mmol, 11%) as a colorless oil. Data obtained
from mixture: IR (neat) 2956, 1614, 1512, 1233, 1080, 923, 758 cm–1; 1H NMR (400
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MHz, CDCl3, signals attributable to [13C]-14 only)  7.12 (2H, d, J = 8.2 Hz), 6.97
(2H, d, J = 8.3 Hz), 5.17 (2H, s), 3.49 (3H, s), 2.32 (3H, d, 1JCH = 126.2 Hz) ppm; 13C
NMR (100 MHz, CDCl3)  155.3 (0), 130.1 (2C, 1), 122.1 (0), 116.5 (2C, 1), 94.9 (2),
56.0 (3), 20.66 (3, enriched) ppm; MS (EI+) m/z 153 (M)+• (54%), 122 (100), 107
13
(28), 92 (73), 78 (67); HRMS (EI+) m/z 153.0869 (calcd. for 12C8 CH12O2:
153.0871).
1-[13C]-Bromomethyl-4-(methoxymethyloxy)benzene ([13C]-15). A stirred solution
of the p-cresol derivative [13C]-14 (1.00 g, 69 wt.% pure, 4.96 mmol) in CCl4 (25 mL)
at rt was treated with N-bromosuccinimide (1.28 g, 7.19 mmol) followed by 2,2´-
azobis(isobutyronitrile) (AIBN, 59 mg, 0.360 mmol). The resulting mixture was
heated to a gentle reflux and stirred for 1.75 h. After this time, the reaction mixture
was allowed to cool, concentrated in vacuo, and the residue purified by column
chromatography (SiO2, eluting with 10% EtOAc in hexanes) to afford a respective
64:36 molar mixture of the desired benzyl bromide [13C]-15 and the corresponding
dibromide (1.04 g, 57 wt.% in [13C]-15, effectively 593 mg, 2.56 mmol, 52%) as a
colorless oil. The benzyl bromide decomposes significantly within 12 hours and it
was used immediately in the next step. Data for [13C]-15: IR (neat) 2957, 2900, 1608,
1511, 1240, 1153, 1080, 838 cm–1; 1H NMR (400 MHz, CDCl3)  7.32 (2H, dd, J =
8.6, 5.0 Hz), 7.01 (2H, d, J = 8.6 Hz), 5.18 (2H, s), 4.49 (2H, d, 1JCH = 153.0 Hz),
3.47 (3H, s) ppm.
1´-[13C]-4-(Methoxymethyloxy)benzaldehyde ([13C]-16). Dimethyl sulfoxide
(DMSO, 8 mL) and solid NaHCO3 (1.30 g, 15.5 mmol) were added to the neat benzyl
bromide [13C]-15 (1.04 g, 57 wt.% pure, 2.56 mmol) and the resulting mixture stirred
under Ar at 90 °C for 2 h. After this time, the mixture was allowed to cool to rt and
partitioned between Et2O (25 mL) and H2O (25 mL). The aqueous phase was
extracted with Et2O (10 mL) and the combined organic phases were washed
successively with H2O (2x10 mL) and brine (10 mL), then dried (Na2SO4), and
concentrated in vacuo. The residue was purified by column chromatography (SiO2,
15% EtOAc in hexanes) to afford the benzaldehyde [13C]-16 (275 mg, 1.65 mmol,
64%) as a colorless oil: IR (neat) 2959, 1652, 1600, 1509, 1242, 1152, 1082, 834
cm–1; 1H NMR (400 MHz, CDCl3)  9.90 (1H, d, 1JCH = 172.6 Hz), 7.84 (2H, dd, J =
8.5, 4.7 Hz), 7.15 (2H, d, J = 8.6 Hz), 5.26 (2H, s), 3.49 (3H, s) ppm; 13C NMR (100
MHz, CDCl3)  191.1 (0, enriched), 162.4 (0), 132.1 (2C, 1), 130.9 (0, d, J = 56 Hz),
116.5 (2C, 1), 94.3 (2), 56.6 (3) ppm; MS (EI+) m/z 167 (M)+• (100%), 152 (65), 122
13
(34); HRMS (EI+) m/z 167.0663 (calcd. for 12C8 CH10O3: 167.0664).
(E)-1,3-[13C]2-2´,4´-Bis(benzyloxymethoxy)-6´-methoxy-4´´-(methoxymethyloxy)-
3´-(3-methylbuten-2-yl)chalcone ([13C]2-17). A stirred solution of the methyl ketone
[13C]-11 (403 mg, 0.821 mmol) and the aryl aldehyde [13C]-16 (247 mg, 1.48 mmol,
1.8 eq) in MeOH (8 mL) was treated with 10 wt.% aq. NaOH (1.00 mL, 2.50 mmol,
3.0 eq) and heated at a gentle reflux for 18 h. After this time, the mixture was
partitioned between EtOAc (30 mL), H2O (30 mL), and brine (10 mL). The aqueous
phase was extracted with EtOAc (2x15 mL) and the combined organic phases were
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washed with brine (15 mL), dried (Na2SO4), and concentrated in vacuo. The residue
was purified by column chromatography (SiO2, eluting with 20% EtOAc in hexanes)
to afford in order of elution recovered aldehyde [13C]-16 (127 mg, 0.76 mmol) and the
title chalcone [13C]2-17 (350 mg, 0.547 mmol, 67%) as a pale yellow oil: IR (neat)
2918, 1596, 1509, 1454, 1240, 1153, 1081, 986, 830, 737 cm–1; 1H NMR (400 MHz,
CDCl3)  7.45 (2H, dd, J = 8.7, 4.6 Hz), 7.36 (1H, ddd, J = 154, 16.0, 6.9 Hz), 7.37-
7.25 (10H, m), 7.02 (2H, d, J = 8.8 Hz), 6.89 (1H, dd, J = 16.1, 2.6 Hz), 6.68 (1H, s),
5.35 (2H, s), 5.22 (1H, tm, J = 6.6 Hz), 5.20 (2H, s), 5.06 (2H, s), 4.74 (2H, s), 4.69
(2H, s), 3.74 (3H, s), 3.48 (3H, s), 3.39 (2H, d, J = 6.7 Hz), 1.76 (3H, s), 1.67 (3H, s)
ppm; 13C NMR (100 MHz, CDCl3)  194.2 (0, enriched), 159.2 (0), 157.9 (0), 156.5
(0), 154.3 (0), 144.2 (1, enriched), 137.5 (0, d, J = 45 Hz), 131.4 (0), 130.2 (2C, 1),
128.7 (2C, 1), 128.5 (2C, 1), 128.21 (2C, 1), 128.15 (1), 127.9 (2C, 1), 127.7 (1),
123.5 (1), 118.5 (0, d, J = 54 Hz), 117.5 (0), 116.6 (2C, 1, d, J = 4 Hz), 98.9 (2), 95.5
(1), 94.4 (2), 92.5 (2), 79.0 (1), 71.7 (2), 70.2 (2), 59.8 (3), 56.3 (3), 25.9 (3), 23.3 (2),
18.1 (3) ppm; MS (ES+) m/z 663 (M+Na)+, 641 (M+H)+; HRMS (ES+) m/z 641.2996
(calcd. for 12C3713C2H43O8: 641.3025).
[13C]2-Xanthohumol = (E)-1,3-[13C]2-6´-methoxy-3´-(3-methylbuten-2-yl)-2´,4´,4´´-
trihydroxychalcone ([13C]2-1). A cloudy solution/suspension of protected
xanthohumol [13C]2-17 (375 mg, 0.586 mmol) in i-PrOH (6 mL) was treated with
conc. aq. HCl (3 drops) and stirred at 60 °C (bath temp.) for 2.66 h (note: the
reaction mixture became an homogenous yellow solution after ca. 10 min). After this
time, the solution was allowed to cool to rt and partitioned between EtOAc (20 mL),
sat. aq. NaHCO3 (10 mL), and H2O (10 mL). The aqueous phase was extracted with
EtOAc (10 mL) and the combined organic phases washed with H2O (10 mL), brine
(10 mL), and then dried (Na2SO4) and concentrated in vacuo. The residue was
subjected to two successive column chromatographic operations (column #1 = SiO2,
eluting with 1.5% MeOH in CH2Cl2; column #2 = SiO2, eluting with 33-50% EtOAc in
hexanes) to obtain pure labelled xanthohumol [13C]2-1 (120 mg, 0.337 mmol, 58%)
as a yellow solid: mp 162-164 °C; IR (ATR) 3178, 2921, 1582, 1538, 1510, 1432,
1331, 1219, 1192, 1167, 1139, 1098, 1052. 971, 823, 802 cm–1; 1H NMR (400 MHz,
CDCl3)  14.68 (1H, s), 7.79 (1H, ddd, J = 15.3, 5.2, 2.4 Hz), 7.75 (1H, ddd, J = 155,
15.5, 6.0 Hz), 7.52 (2H, dd, J = 8.2, 4.3 Hz), 6.86 (2H, d, J = 8.4 Hz), 6.19 (1H, s),
5.95 (1H, s), 5.30 (1H, tm, J = 7.5 Hz), 5.06 (1H, s), 3.90 (3H, s), 3.41 (2H, d, J = 7.1
Hz), 1.83 (3H, s), 1.78 (3H, s) ppm; 1H NMR (400 MHz, d6-DMSO)  14.64 (1H, s),
10.56 (1H, s), 10.06 (1H, s), 7.76 (1H, dm, J = 15.6 Hz), 7.67 (1H, ddd, J = 156,
15.6, 5.6 Hz), 7.57 (2H, dd, J = 7.8, 4.9 Hz), 6.84 (2H, d, J = 8.1 Hz), 6.08 (1H, s),
5.14 (1H, br t, J = 7.0 Hz), 3.87 (3H, s), 3.13 (2H, d, J = 7.1 Hz), 1.70 (3H, s), 1.61
(3H, s) ppm; 13C NMR (175 MHz, d6-DMSO)  191.7 (0, enriched), 164.6 (0), 162.4
(0), 160.5 (0), 159.9 (0), 142.5 (1, enriched), 130.5 (2C, 1), 129.9 (0), 126.0 (0, dd, J
= 58, 7 Hz), 123.7 (1, dd, J = 68, 54 Hz), 123.0 (1), 115.9 (2C, 1, d, J = 4 Hz), 107.3
(0), 104.5 (0, d, J = 61 Hz), 90.9 (1), 55.7 (3), 25.5 (3), 21.0 (2), 17.6 (3) ppm; MS
(ES+) m/z 357 (M+H)+; HRMS (ES+) m/z 357.1630 (calcd. for 12C1913C2H23O5:
357.1613).
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A sample of commercially available natural xanthohumol (1) gave mp = 162-165 °C
on the same melting point apparatus and exhibited 1H and 13C NMR spectral
signatures that differed only as expected from those obtained for synthetic [13C]2-1.
Data for xanthohumol (1): IR (ATR) 3177, 2914, 1599, 1429, 1340, 1291, 1219,
1166, 1098, 973, 868, 798 cm–1; 1H NMR (400 MHz, CDCl3)  14.66 (1H, s), 7.80
(1H, d, J = 15.4 Hz), 7.75 (1H, d, J = 15.6 Hz), 7.52 (2H, d, J = 8.6 Hz), 6.86 (2H, d,
J = 8.6 Hz), 6.17 (1H, s), 5.94 (1H, s), 5.29 (1H, tm, J = 7.2 Hz), 5.03 (1H, s), 3.90
(3H, s), 3.41 (2H, d, J = 7.5 Hz), 1.83 (3H, s), 1.78 (3H, s) ppm; 1H NMR (400 MHz,
d6-DMSO)  14.61 (1H, s), 10.52 (1H, s), 10.03 (1H, s), 7.76 (1H, d, J = 15.6 Hz),
7.67 (1H, J = 15.5 Hz), 7.57 (2H, d, J = 8.6 Hz), 6.84 (2H, d, J = 8.6 Hz), 6.09 (1H,
s), 5.14 (1H, tm, J = 7.2 Hz), 3.87 (3H, s), 3.14 (2H, d, J = 7.0 Hz), 1.70 (3H, s), 1.61
(3H, s) ppm; 13C NMR (100 MHz, d6-DMSO)  191.8 (0), 164.7 (0), 162.5 (0), 160.6
(0), 160.0 (0), 142.6 (1), 130.6 (2C, 1), 130.0 (0), 126.1 (0), 123.9 (1), 123.1 (1),
116.1 (2C, 1), 107.4 (0), 104.7 (0), 91.0 (1), 55.8 (3), 25.5 (3), 21.1 (2), 17.7 (3) ppm.
Note: If present in synthetic [13C]2-1, contamination from traces of HCl addition
adduct [13C]2-18 is most obviously revealed in the 1H NMR spectrum by an AA´BB´
system for ArCH2CH2C(Cl)Me2 at H (400 MHz, CDCl3) = 2.81 (2H, AA´BB´ width
16.3 Hz), 1.96 (2H, AA´BB´ width 16.2 Hz) ppm. The purification protocol described
above will ensure that homogenous samples of [13C]2-1 completely free of [13C]2-18
are obtained.
[13C]3-Xanthohumol = (E)-1,3-[13C]2-6´-[13C]methoxy-3´-(3-methylbuten-2-yl)-
2´,4´,4´´-trihydroxychalcone ([13C]3-1). Prepared by analogy to the double-labelled
isotopomer of xanthohumol ([13C]2-1) from the corresponding triple-labelled [13C]3-17.
Data for [13C]3-1: mp 162-165 °C; IR (ATR) 3178, 2918, 1588, 1575, 1510, 1469,
1436, 1330, 1190, 1167, 1134, 1095, 1050, 970, 821, 798 cm–1; 1H NMR (400 MHz,
CDCl3)  14.63 (1H, s), 7.79 (1H, ddd, J = 15.4, 5.1, 2.2 Hz), 7.76 (1H, ddd, J = 157,
15.3, 6.2 Hz), 7.52 (2H, dd, J = 7.8, 4.9 Hz), 6.86 (2H, d, J = 8.6 Hz), 6.15 (1H, br s),
5.94 (1H, s), 5.30 (1H, tm, J = 7.6 Hz), 4.98 (1H, br s), 3.90 (3H, d, J = 145 Hz), 3.41
(2H, d, J = 6.9 Hz), 1.83 (3H, s), 1.78 (3H, s) ppm; 1H NMR (700 MHz, d6-DMSO,
spectral width limited to 10 to –1 ppm, therefore OH signals not observed)  7.76
(1H, ddd, J = 15.2, 8.9, 5.3 Hz), 7.67 (1H, ddd, J = 158, 15.2, 6.1 ppm), 7.57 (2H, dd,
J = 8.6, 4.3 Hz), 6.83 (2H, d, J = 8.6 Hz), 6.08 (1H, s), 5.14 (1H, tm, J = 7.1 Hz), 3.86
(3H, d, J = 146 Hz), 3.14 (2H, d, J = 7.1 Hz), 1.70 (3H, s), 1.61 (3H, s) ppm; 13C
NMR (175 MHz, d6-DMSO)  191.6 (0, enriched), 164.6 (0), 162.4 (0), 160.5 (0),
159.9 (0), 142.5 (1, enriched), 130.5 (2C, 1, d, J = 3.5 Hz), 129.9 (0), 126.1 (0, d, J =
52 Hz), 123.7 (1, dd, J = 68, 54 Hz), 123.0 (1), 115.9 (2C, 1, d, J = 4 Hz), 107.3 (0),
104.5 (0, d, J = 60 Hz), 90.9 (1), 55.7 (3, enriched), 25.5 (3), 21.0 (2), 17.6 (3) ppm.
MS (ES+) m/z 358 (M+H)+; HRMS (ES+) m/z 358.1634 (calcd. for 12C1813C3H23O5:
358.1646).
[13C]5-Xanthohumol = (E)-1,3-[13C]2-6´-[13C]methoxy-3´-(1,2-[13C]2-3-
methylbuten-2-yl)-2´,4´,4´´-trihydroxychalcone ([13C]5-1). Prepared by analogy to
the double-labelled isotopomer of xanthohumol ([13C]2-1) from the corresponding
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penta-labelled [13C]5-17. Data for [13C]5-1: 1H NMR (400 MHz, CDCl3)  14.67 (1H,
s), 7.79 (1H, ddd, J = 15.0, 5.0, 2.2 Hz), 7.76 (1H, ddd, J = 155, 15.0, 5.8 Hz), 7.52
(2H, dd, J = 8.3, 4.2 Hz), 6.87 (2H, d, J = 8.5 Hz), 6.20 (1H, br s), 5.95 (1H, s), 5.30
(1H, dm, J = 151 Hz), 5.10 (1H, br s), 3.90 (3H, d, J = 145 Hz), 3.40 (2H, dt, J = 129,
7.1 Hz), 1.83 (3H, d, J = 4.9 Hz), 1.78 (3H, d, J = 6.0 Hz) ppm; 1H NMR (700 MHz,
d6-DMSO)  14.63 (1H, s), 10.55 (1H, br s), 10.05 (1H, br s), 7.77 (1H, ddd, J = 15.2,
8.9, 5.5 Hz Hz), 7.67 (1H, ddd, J = 155, 15.4, 6.3 Hz), 7.57 (2H, dd, J = 8.6, 4.3 Hz),
6.84 (2H, d, J = 8.6 Hz), 6.08 (1H, s), 5.13 (1H, dm, J = 152 Hz), 3.86 (3H, d, J = 145
Hz), 3.13 (2H, dt, J = 128, 6.8 Hz), 1.70 (3H, d, J = 4.9 Hz), 1.60 (3H, d, J = 5.8 Hz)
ppm; 13C NMR (175 MHz, d6-DMSO)  191.5 (0, enriched), 164.6 (0), 162.4 (0),
160.5 (0), 159.9 (0), 142.5 (1, enriched), 130.5 (2C, 1), 129.9 (0, d, J = 74 Hz), 126.0
(0, dm, J = 63 Hz), 123.7 (1, dd, J = 70, 56 Hz), 123.0 (1, d, J = 44 Hz, enriched),
116.0 (2C, 1), 107.3 (0, d, J = 46 Hz), 104.5 (0, d, J = 58 Hz), 90.9 (1), 55.8 (3,
enriched), 25.5 (3), 21.0 (2, d, J = 44 Hz), 17.7 (3) ppm; MS (ES+) m/z 360 (M+H)+;
HRMS (ES+) m/z 360.1723 (calcd. for 12C1613C5H23O5: 360.1713).
Conclusion
The Khupse and Erhardt total synthesis of xanthohumol was re-evaluated and
modified to incorporate readily available 13C-labelled starting materials to enable the
preparation of [13C]2-, [13C]3-, and [13C]5-isotopomers of this prenylated flavonoid
natural product. The labelled xanthohumols were obtained in ca. 6% overall yield via
a synthetic route involving a 7 step longest linear sequence from phloroglucinol with
1-[13C]-acetyl chloride, [13C]-iodomethane, and ethyl 1,2-[13C]2-bromoacetate as
sources of 13C atoms. The labelled compounds prepared will be useful in
experiments to probe the biological activity of xanthohumol and its metabolism in
vivo.
Acknowledgements
This work was supported by a grant from the National Center for Complementary
and Integrated Health at the National Institutes of Health (5R01AT009168 to AFG,
JFS and CSM). Donovon A. Adpressa and Stephen Huhn are thanked for their
technical assistance with the collection of NMR spectra of 13C-labelled
xanthohumols. Jeffrey T. Morré (OSU Mass Spectrometry Center) is likewise
gratefully acknowledged for help with the collection of mass spectra.
Conflict of Interest
The authors did not report any conflict of interest.
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References
1. a) J. F. Stevens, J. E. Page, Phytochem. 2004, 65, 1317-1330; b) J. F.
Stevens, C. S. Maier, Phytochem. Rev. 2016, 15, 425-444.
2. a) S. Venturelli, M. Burkard, M. Biendl, U. M. Lauer, J. Frank, C. Busch,
Nutrition 2016, 32, 1171-1178; b) C. Gerhaeuser, Eur. J. Cancer 2005, 41,
1941-1954.
3. a) R. Costa, I. Rodrigues, L. Guardao, S. Rocha-Rodrigues, C. Silva, J.
Magalhaes, M. Ferreira-de-Almeida, R. Negrao, R. Soares, J. Nutri. Biochem.
2017, 45, 39-47; b) C. L. Miranda, V. D. Elias, J. J. Hay, J. Choi, R. L. Reed, J.
F. Stevens, Arch. Biochem. Biophys. 2016, 599, 22-30; c) L. Legette, C.
Karnpracha, R. L. Reed, J. Choi, G. Bobe, J. M. Christensen, R. Rodriguez-
Proteau, J. Q. Purnell, J. F. Stevens, Mol. Nutri. Food Res. 2014, 58, 248-255;
d) J. S. Kirkwood, L. C. L. Legette, C. L. Miranda, Y. Jiang, J. F. Stevens, J.
Biol. Chem. 2013, 288, 19000-19013; e) L. C. L. Legette, A. Y. Moreno Luna,
R. L. Reed, C. L. Miranda, G. Bobe, R. R. Proteau, J. F. Stevens, Phytochem.
2013, 91, 236-241.
4. a) P. Cos, T. De Bruyne, S. Apers, D. Vanden Berghe, L. Pieters, V. Luc, A. J.
Vlietinck, Planta Medica 2003, 69, 589-599; b) S. R. Milligan, J. C. Kalita, A.
Heyerick, H. Rong, L. De Cooman, D. De Keukeleire, J. Clin. Endocrinol.
Metabol. 1999, 84, 2249-2252.
5. M. Vandewalle, Bull. Soc. Chim. Belg. 1961, 70, 163-167.
6. R. S. Khupse, P. W. Erhardt, J. Nat. Prod. 2007, 70, 1507-1509.
7. A synthesis of desmethylxanthohumol has also been reported, see: R. A. Diller,
H. M. Riepl, O. Rose, C. Frias, G. Henze, A. Prokop, Chem. Biodiversity 2005,
2, 1331-1337.
8. B. Zhang, D. Duan, C. Ge, J. Yao, Y. Liu, X. Li, J. Fang, J. Med. Chem. 2015,
58, 1795-1805.
9. Presented in part at the 253rd ACS National Meeting & Exposition, San
Francisco, CA, USA, April 2-6, 2017: D. Ellinwood, P. R. Blakemore Abstracts
of Papers, American Chemical Society: Washington, D.C., 2017; paper CHED-
1429.
10. M. Morkunas, L. Dube, F. Götz, M. E. Maier, Tetrahedron 2013, 69, 8559-8563.
11. E. D. Burling, A. Jefferson, F. Scheinmann, Tetrahedron 1965, 21, 2653-2669.
12. X. Bu, L. Zhao, Y. Li, Synthesis 1997, 1246-1248.
13. a) S. Gester, P. Metz, O. Zierau, G. Vollmer, Tetrahedron 2001, 57, 1015-1018;
b) B. M. Trost, F. D. Toste, J. Am. Chem. Soc. 1998, 120, 815-816; c) K. H.
Almabruk, J. H. Chang, T. Mahmud, J. Nat. Prod. 2016, 79, 2391-2396.
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14. For representative examples, see: a) G. Büchi, J. C. Leung, J. Org. Chem.
1986, 51, 4813-4818; b) L. M. Harwood, A. J. Oxford, C. Thomson, J. Chem.
Soc., Chem. Commun. 1987, 1615-1617; c) N. Cairns, L. M. Harwood, D. P.
Astles, J. Chem. Soc., Perkin Trans. 1 1994, 3101-3107; d) R. E. Patre, J. B.
Shet, P. S. Parameswaran, S. G. Tilve, Tetrahedron Lett. 2009, 50, 6488-6490.
15. H. Wang, Z. Yan, Y. Lei, K. Sheng, Q. Yao, K. Lu, P. Yu, Tetrahedron Lett.
2014, 55, 897-899.
16. K. C. Nicolaou, P. S. Baran, Y.-L. Zhong, J. Am. Chem. Soc. 2001, 123, 3183-
3185.
17. C. Jin, L. Zhang, W. Su, Synlett 2011, 1435-1438.
18. C. Djerassi, Chem. Rev. 1948, 43, 271-317.
19. N. Kornblum, W. J. Jones, G. J. Anderson, J. Am. Chem. Soc. 1959, 81, 4113-
4114.
20. D. G. Hall, P. Deslongchamps, J. Org. Chem. 1995, 60, 7796-7814.
21. P. B. Shrestha-Dawadi, J. Lugtenburg, Eur. J. Org. Chem. 2003, 4654-4663.
22. M. E. Hart, K. L. Suchland, M. Miyakawa, J. R. Bunzow, D. K. Grandy, T. S.
Scanlan, J. Med. Chem. 2006, 49, 1101-1112.
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Scheme 1. Synthesis of methyl ketone fragment [13C]-11.  = 13C; BOM =
benzyloxymethyl; DEAD = diethyl azodicarboxylate; fod = 6,6,7,7,8,8,8-
heptafluoro-2,2-dimethyl-3,5-octanedionato.
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Scheme 2. Synthesis of aldehyde fragment [13C]-16.  = 13C; MOM =
methoxymethyl; NBS = N-bromosuccinimide; AIBN = 2,2´-azobis(isobutyronitrile).
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O
BOMO
O
aq. NaOH
H
+
*
*
MeOH, D
18 h, 67%
BOMO
OCH3
OMOM
[13C]-16
[13C]-11
BOM
O
c. HCl
O
*
*
i-PrOH, 60 °C
2.7 h
BOMO
OCH3
OMOM
Cl
[13C]2-17
OH O
OH O
+
*
* Ar
*
*
HO
OCH3
OH
HO
OCH3
1,3-[13C]2-xanthohumol ([13C]2-1)
[13C]2-18, 1-2%
58%
Ar = 4-(HO)C6H4
Scheme 3. Completion of synthesis of 1,3-[13C]2-xanthohumol ([13C]2-1).  = 13C; BOM
= benzyloxymethyl; MOM = methoxymethyl.
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d
H = 14.7 ppm
H
1´
O
O
xanthohumol (1)
HO
OCH3
OH
OH O
3
enol/orthoquinomethide tautomer
3´
1
4´´
6´
HO
OCH3
OH
PG
O
O
O
+
H
O PG´
PG O
OCH3
2
3
Figure 1. Xanthohumol (1) and the classical retrosynthetic disconnection for chalcone
synthesis via aldol condensation of a methyl ketone (2) and an aryl aldehyde (3).
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d
H2 = 7.79 ppm, ddd, J = 15.3, 5.2, 2.4 Hz
O
H
H
2´´
1
3
dH3 = 7.75 ppm, ddd, J = 155, 15.5, 6.0 Hz
dH2´´ = 7.52 ppm, dd, J = 8.2, 4.3 Hz
Ar´
*
*
H
H
OH
[13C]2-1
dH2
´´
dH2 dH3
Figure 2. Excerpt of 1H NMR (400 MHz, CDCl3) spectrum for 1,3-[13C]2-xanthohumol
([13C]2-1) showing effects of J-coupling to 13C-atoms at C1 and C3 by H2, H3, and
H2´´. Coupling constants due to interactions with 13C atoms are underlined.  = 13C.
*
OH O
OH O
*
* Ar
* Ar
*
*
*
*
HO
OCH3
HO
OCH3
[13C]3-xanthohumol ([13C]3-1)
[13C]5-xanthohumol ([13C]5-1)
Figure 3. Two additional [13C]-labelled isotopomers of xanthohumol that were
prepared by analogy to [13C]2-1. Ar = 4-(HO)-C6H4.
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