A Condensed, Scalable Synthesis of Racemic Koningic Acid

Article abstract of DOI:10.1021/acs.joc.0c00344

The natural product koningic acid (KA) is a selective covalent inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a critical node in the glycolysis pathway. While KA is available commercially, sources are limited and its cost becomes rapidly prohibitive beyond the milligram scale. Additionally, a practical and flexible synthetic route to KA and analogs remains to be developed. Here we detail a new route that is operationally safer, scalable and offers a five-step reduction in the previously reported longest linear sequence.

Full text of DOI:10.1021/acs.joc.0c00344

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Note
A Condensed, Scalable Synthesis of Racemic Koningic Acid
Guillaume Barbe, David Chai, Bin Chen, Daniel Guay,
Eric Levesque, John Mancuso, and Brian DeChristopher
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c00344 • Publication Date (Web): 20 Apr 2020
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A Condensed, Scalable Synthesis of Racemic
Koningic Acid
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Guillaume Barbe,1 David Chai,2 Bin Chen,2 Daniel Guay,2 Eric Levesque,2 John Mancuso,2 and
Brian DeChristopher1*
1. Rheos Medicines Inc. 38 Sidney St. Suite 200, Cambridge, MA 02139
2. NuChem Therapeutics Inc. 2350 Cohen, Suite 201, Saint-Laurent, QC Canada, H4R 2N6
KEYWORDS Koningic acid, heptelidic acid, glycolysis inhibitor, total synthesis
bdechristopher@rheosrx.com
The natural product koningic acid (KA) is a selective covalent inhibitor of glyceraldehyde-
3-phosphate dehydrogenase (GAPDH), a critical node in the glycolysis pathway. While KA
is available commercially, sources are limited and its cost becomes rapidly prohibitive
beyond the milligram scale. Additionally, a practical and flexible synthetic route to KA
and analogs remains to be developed. Here we detail a new route that is operationally
safer, scalable and offers a five step reduction in the previously reported longest linear
sequence.
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Glycolysis is a cornerstone metabolic pathway for cellular energy supply that involves the
conversion of glucose into pyruvate, ATP and NADH. The pathway becomes critically important
when cells are unable to produce energy by other means, such as in a hypoxic environment (i.e.
anaerobic glycolysis) or when the energy demand surges (e.g. immune cell activation, rapid cell
proliferation) under normoxic conditions (i.e. aerobic glycolysis).1 The latter phenomenon, known
as the Warburg effect, enables cell proliferation (e.g. cancer and immune cells) by generating
energy and essential biosynthetic building blocks from glycolytic intermediates to permit growth
and duplication of cellular components during division.2
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As part of our effort to gain a high-resolution understanding of the role metabolism plays
in immune cell function, we became interested in opportunities to pharmacologically interrogate
glycolysis. One such opportunity is the natural product koningic acid (KA, 1, Figure 1), a potent,
selective and well-characterized covalent inhibitor of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH).3 This enzyme catalyzes the conversion of glyceraldehyde-3-phosphate (G3P) to D-
glycerate 1,3-bisphosphate (1,3BPG) (Figure 1) and resides at the interface between the ATP-
consuming (-2 ATP) and the ATP-producing (+4 ATP) phases of glycolysis. Taken together, the
critical role of GAPDH and the strength of KA as a chemical tool provided a unique opportunity
to study the role of glycolysis in immune cells.4
Figure 1. GAPDH inhibitor koningic acid (1).
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O
H
H
O
O
HO
O
koningic acid (1)
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O
O
GAPDH
2-O3PO
H
2-O3PO
OPO3
2-
OH
G3P
OH
1,3BPG
NAD+
NADH
While KA is commercially available via microbial fermentation from Trichoderma fungus,
sources are limited and the cost of multi-milligram quantities becomes rapidly prohibitive. These
factors limit the ability to pharmacologically interrogate GAPDH as an important node of
glycolysis as well as to access synthetically-derived KA analogs or tools (e.g. inactive controls,
molecular probes, etc.). Given these limitations, we initiated an effort aimed at developing a
synthesis that offered rapid, reliable access to useful quantities of KA and its analogs.
In 1988, Danishefsky reported the first total synthesis of racemic KA (rac-KA).5 In 1997,
Urban adapted Danishefsky’s synthesis into a chiral auxiliary-based, asymmetric formal synthesis
of the natural product.6 To first access KA, we used Danishefsky’s synthesis of rac-KA followed
by chiral separation into its natural and non-natural enantiomers and were able to secure milligram
quantities (data not shown). As we progressed through the synthesis, however, multiple
opportunities were identified for improvement. Herein, we report a condensed and scalable
synthesis of racemic KA.
A key step in Danishefsky’s synthesis is a conjugate addition reaction that brings together
14 of the 15 carbons present in KA (Scheme 1). While the chemistry worked well in our hands,
we sought improvements in several areas. First, we found that the Michael acceptor 2 could readily
undergo an irreversible tautomerization to a corresponding dienol upon storage. Since the dienol
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is completely unreactive under the conjugate addition conditions, we found it imperative to use
the material right away in the next step. Second, the protected vinyl bromide 3 was produced in
56% yield over five steps and required storage at -20 °C to minimize spontaneous TBS
deprotection, which was occasionally observed over time. As such, we saw an opportunity to
reduce the number of steps while increasing the overall yield and practicality of the synthesis.
Third, we were interested in finding an alternative to the use of tBuLi as a lithiation agent in order
to improve the overall safety profile of the synthetic route upon scale up.
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Scheme 1. Circumventing the use of tBuLi in the synthesis.
TBSO
TBSO
Br
3
tBuLi, CuI, Et2O
O
O
O
OH
Original work
MeO
MeO
RO
This work
RO
Li
CuCN
S
2
5a
5b
R=TBS
R=
nBuLi, Et2O, THF
O
I
O
4
66% yield
Inspired by the work of Urban,6 we turned our attention to vinyl iodide 4, which was found
to offer multiple advantages. First, 4 was readily available on multi-gram scale in 3 steps from
commercially available starting materials. Second, the acetonide protecting group was stable upon
long-term storage (vs TBS, see above). Third, it was hypothesized that a more reactive vinyl iodide
(vs bromide) could enable the substitution of tBuLi with a safer alternative. Gratifyingly, lithium-
halogen exchange with nBuLi was successful and the subsequent transmetallation with copper
enabled the desired 1,4-addition to enone ester 2 to afford 5b as a keto/enol mixture. While having
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a mixture is inconsequential to the next step (see below), it did complicate the purification
procedure upon scale up. Indeed, when conducted on a relatively small scale (e.g. 1-2 g),
purification by flash chromatography was uneventful and provided pure material 5b in 66% yield.
However, upon scaling the reaction to over 20 g of the Michael acceptor 2, purification by flash
chromatography was complicated by the ability of 5b to tautomerize on silica gel. To circumvent
this issue, crystallization conditions were developed for isolation of 5b, albeit with a decreased
27% yield. Interestingly, the keto tautomer preferentially crystallized from the mixture and the
isolated material contained only trace amounts of the enol form. Overall, the development of this
conjugate addition procedure alleviated the scale limitations imposed by the original use of tBuLi
as well as long term stability concerns for TBS-protected intermediates. Moreover, initial
crystallization work provided a promising starting point for future efforts to streamline
purification.
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The next critical component of the synthesis of KA involves the introduction of the final
carbon that will host the epoxide functionality. Originally, this was achieved via a 4-step sequence
that featured nickel cross-coupling and allylsilane protodesilylation reactions (Scheme 2). We
envisioned that, theoretically, a Wittig reaction could directly provide us with the desired exocyclic
alkene 9. Though we initially suspected that the acidity of the hydrogen atom residing between the
two carbonyls might simply quench the Wittig reagent, a number of similar cases were previously
reported in the literature.7 Initial investigation into the Wittig olefination led to two key
observations (Table 1). First, when preparing the Wittig reagent in situ, the conversion of the
olefination reaction is strongly dependent on the nature of the cationic counterion (Table 1, entries
1-4).8 Additionally, the nature of the reaction product (i.e. desired 9 vs. isomerized products 10) is
significantly impacted by the number of equivalents of the Wittig reagents (Table 1, entry 5).
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Based on the latter observation, it was postulated that use of the free ylide would be an efficient
way to prevent any excess basic species in the reaction. We were pleased to find that upon using
the free ylide, the reaction conversion could be increased to 89% while maintaining the integrity
of the resulting double bond (Table 1, entries 6-9). Interestingly, the results with the free ylide
demonstrate that the cationic counterion, while impactful to the reaction outcome, is not necessary
to achieve high conversion. Finally, it is noteworthy that the excess ylide and corresponding
phosphine oxide byproduct could be removed by precipitation in heptane at 0 °C, thereby allowing
for simpler subsequent purification by silica gel chromatography. Under the optimal conditions,
olefin 9 could be obtained in 82% yield, including a minor diastereomer observed by NMR (12:1
ratio favoring 9).
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Scheme 2. Introduction of the final carbon atom of KA.
O
P
Original work
This work
MeO
O
OEt
OH
O
O
OH
O
OEt
MeO
MeO
(EtO)2POCl,
NaH, THF
O
TBSO
TBSO
0 °C to rt
74%
O
TBSO
TBSO
5b
5a
6
TMSCH2MgCl,
Ni(acac)2, THF,
86%
Ph3PCH2
THF
82%
SiMe3
O
O
O
MeO
O
MeO
AcO
MeO
1. FeCl3, Ac2O
2. TFA, DCM
TBSO
O
62% (two steps)
AcO
TBSO
9
8
7
Table 1. Optimization of Wittig reaction conditions.
O
O
O
O
Reagents
O
O
O
O
O
O
O
O
O
+
THF, 60 o
time
C
5b
9
10
Entry
Base
PPh3MeBr
PPh3CH2
Time
Conversion Product
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(equivalents)
LiHMDS (1.9)
NaHMDS (1.9)
KHMDS (1.9)
KOtBu (1.9)
KOtBu (1.05)
(equivalents)
(equivalents)
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2.0
2.0
2.0
2.0
1.1
-
-
-
-
-
-
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-
-
24 h
24 h
24 h
24 h
24 h
20 h
20 h
20 h
20 h
0%
0%
-
-
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9
9
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100%
100%
80%
75%
87%
89%
100%
-
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-
-
1.0
1.1
1.2
1.5
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With the exocyclic double bond in place, we proceeded to complete the total synthesis of
rac-KA (Scheme 3). First, we developed a one-pot, two-step hydrolysis protocol that allowed for
the synthesis of the diol acid 11 in 92% yield. Using the lactonization conditions previously
reported by Danishefsky, followed by a simple extraction procedure and the removal of any
remaining triethylammonium salts by precipitation with acetone, 12 was obtained in sufficient
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purity to be progressed to the next step. We then elected to use MnO2 for the allylic oxidation.
These reaction conditions provided the aldehyde 13 in 66% yield over two steps, results that were
found to be more reproducible than when using Jones oxidation conditions.5 Likewise, we also
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5
found that conducting the subsequent epoxidation reaction using MoO3•H2O2 versus Mo(CO)6 as
a catalyst provided a more reproducible 65% yield of KA precursor 14. Finally, generation of the
carboxylic acid using Pinnick oxidation conditions10 followed by crystallization afforded pure rac-
KA in 87% yield.
Scheme 3. Completion of the synthesis of racemic KA
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O
O
O
i) HCl, H2O
ii) NaOH, H2O
BOP-Cl
DMAP, Et3N
O
MeO
HO
O
HO
HO
DCM
92% over 2 steps
HO
O
9
11
12
9
MnO2, DCM
66% over 2 steps
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O
O
O
H
H
O
O
O
O
O
O
TBHP, MoO3 H2O2
NaOCl, NaH2PO4
tBuOH:H2O
Toluene
65%
O
O
HO
87%
14
13
rac-koningic acid
In summary, a shortened synthesis of racemic KA has been developed which builds on
the foundational work of both the Danishefsky and Urban groups. This new route offers a 5-step
reduction in the longest linear sequence (7.8% overall yield) and alleviates the scale limitations
imposed by the original use of tBuLi for the key conjugate addition step. We believe the
improved practicality of this new route will allow for an improved understanding of structure-
activity relationships around koningic acid and will provide new avenues to interrogate the role
of glycolysis in both immune and non-immune cell function.
Experimental Section
General Information
Unless otherwise noted, all commercial-grade reagents and substrates were ordered from
Sigma Aldrich, TCI America, and Combi-blocks. These chemicals were used without further
purification. Anhydrous solvents were used as received. Reactions were heated using an oil bath.
Column chromatography was performed using Teledyne ISCO Combiflash Rf+ with Redisep Rf
gold silica gel normal phase columns or Biotage SNAP C18 RP columns. Thin-layer
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chromatography (TLC) was performed using glass plates pre-coated with 0.25 mm of silica gel 60
F254. 1H NMR spectra were recorded on either a 300 or 400 MHz NMR spectrometer. Chemical
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shifts (δ values) for H NMR spectra are reported in parts per million (ppm) downfield, and the
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coupling constants (J values) are in Hz. C NMR spectra were recorded on a 100 MHz NMR
spectrometer with complete proton decoupling. LCMS data were acquired on an Agilent single
quadrupole LCMS series 6100.
Methyl 3-isopropyl-6-oxocyclohex-1-ene-1-carboxylate (2)
2 was prepared according to the previously described methods.5
Synthesis of 5-(iodomethylene)-2,2-dimethyl-1,3-dioxane (4)
(iodomethyl)triphenylphosphonium iodide11
A solution of triphenylphosphine (146 g, 556 mmol) and diiodomethane (60.2 mL, 750
mmol) in toluene (487 mL) in a flask equipped with a reflux condenser was heated at 100 °C for
20 h under an argon atmosphere (protected from light by aluminum foil). The reaction mixture
was cooled to 0 °C. The resulting pale white solid was filtered, washed with cold toluene and
CH2Cl2 and then dried under vacuum to give (iodomethyl)triphenylphosphonium iodide (287 g,
542 mmol, 97.6% yield) as a pale white solid. LCMS m/z: [M-I]+ Calcd for C19H17IP 403.0; Found
403.0. 1H NMR (DMSO-d6, 400 MHz): δH 7.91-7.74 (15H, m), 5.03 (2H, d, J = 8.7 Hz).
2,2-dimethyl-1,3-dioxan-5-one12
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Tris(hydroxylmethyl)aminomethane hydrochloride (100 g, 634 mmol) and 4-
methylbenzene-1-sulfonic acid monohydrate (5.42 g, 28.5 mmol) were added to dry
dimethylformamide (257 mL). 2,2-dimethoxypropane (85.2 mL, 697 mmol) was added and the
mixture was stirred at rt for 60 h. Triethylamine (14.8 mL, 107 mmol) was added and the reaction
mixture was concentrated under reduced pressure. The resulting crude gel was sonicated in MeOH
(1 L). The solvent was removed to give a white solid mixture. The residue was suspended in ethyl
acetate (860 mL) and triethylamine (143 mL, 1.03 mol) and the mixture was stirred at rt for 10
min. The precipitate was filtered off and the solvents were removed under reduced pressure to give
crude (5-amino-2,2-dimethyl-1,3-dioxan-5-yl)methanol as a yellow oil (97.68 g, 545 mmol
product (86.0% yield) calculated by 1H NMR). No mass found by LCMS. 1H NMR (CDCl3, 400
MHz): δH 3.79 (2H, d, J = 11.8 Hz), 3.55 (2H, d, J = 11.7 Hz), 3.50 (2H, s), 2.46 (3H, br s), 1.44
(3H, s), 1.41 (3H, s).
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A mixture of sodium periodate (151 g, 708 mmol) in water (1.22 L) was added over 90
min to the crude (5-amino-2,2-dimethyl-1,3-dioxan-5-yl)methanol (97.68 g, 545 mmol) in water
(700 mL) and methanol (180 mL) at 0 °C. The mixture was stirred for 1.5 h at 0 °C. The white
suspension was filtered off and the solution was extracted with CH2Cl2 (x7). The combined organic
layers were washed with a sodium bicarbonate solution (5%; x2), dried over sodium sulfate and
evaporated under reduced pressure (keeping the temperature below 20 °C). The crude was purified
by vacuum distillation at 15 mbar using an oil bath at 110 °C, with fractions collected at 55-60 °C.
Portion A (beginning), 8.2 g, colorless oil, contained 7.19 g pure product 2,2-dimethyl-1,3-dioxan-
5-one, 12% w/w DMF; portion B, 38.95 g, colorless oil, contained 36.88 g pure product, 5% w/w
DMF. Total amount of product 2,2-dimethyl-1,3-dioxan-5-one was 44.07 g (339 mmol, 53% yield
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in two steps). No mass found by LCMS. Rf = 0.4 (20% EtOAc/hexane). H NMR (CDCl3, 400
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MHz): δH 4.15 (4H, s), 1.45 (6H, s).
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5-(iodomethylene)-2,2-dimethyl-1,3-dioxane (4)
To a suspension of (iodomethyl)triphenylphosphonium iodide (164 g, 311 mmol) in
tetrahydrofuran (725 mL) at -23 °C was slowly added sodium bis(trimethylsilyl)amide (311 mL,
311 mmol) (1M in THF). After stirring for 15 min, 2,2-dimethyl-1,3-dioxan-5-one (38.95 g, 283
mmol) was added. The reaction mixture was allowed to warm to rt and was stirred for 1 h. Water
(100 mL) was added to the mixture, the suspension was filtered and the filtrate was extracted with
MTBE (3 × 800 mL). The combined organic layers were dried over anhydrous sodium sulfate and
concentrated under reduced pressure to obtain the crude product. 250 g silica gel and 300 mL DCM
were added to the crude residue. The mixture was dried under reduced pressure to give a free-
flowing powder. Filter paper was placed over a sand core funnel (dm ~ 13 cm), to which silica gel
(~ 1 cm) was added. The silica gel was washed with hexanes under vacuum. The crude dry powder
was then loaded in the funnel. A filter paper was put on the top of the powder. This solid was
washed with hexanes (1500 mL) under vacuum. The filtrate was collected in 4 portions. Product
1
was detected via TLC in the first three portions. Each portion was concentrated and H NMR
showed clean desired product. The three portions were combined to give 4 (56.9 g, 223 mmol,
79.1% yield) as a brown oil containing 2% mol/mol of triphenylphosphine oxide. HRMS (ESI)
m/z: [M+Na]+ Calcd for C7H11IO2Na 276.9701; Found 276.9686. Rf = 0.5 (20% EtOAc/hexane).
IR (Nujol) νmax: 2986, 2846, 1632, 1445, 1380, 1370, 1277, 1236, 1216, 1194, 1120, 1083, 1035,
1017, 828, 765, 731, 680, 644, 541, 516 cm–1. 1H NMR (CDCl3, 400 MHz): δH 5.98 (1H, m), 4.27-
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4.26 (2H, m), 4.22 (2H, s), 1.39 (6H, s). 13C NMR (CDCl3, 100 MHz): δC 145.0, 99.7, 71.8, 66.3,
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Methyl (2SR,3RS)-2-((2,2-dimethyl-1,3-dioxan-5-ylidene)methyl)-3-isopropyl-6-oxocyclohexane-
1-carboxylate (5b)6
A solution of 4 (1.96 g, 7.73 mmol) in diethyl ether (30 mL) was cooled to -78 °C. n-
Butyllithium (3.7 mL, 9.26 mmol, 2.5 M solution in hexane) was added and the mixture was stirred
for 45 minutes at -78 °C. Subsequently, this mixture at -78 °C was cannulated dropwise into a
stirred solution of lithium 2-thienylcyanocuprate (31.3 mL, 7.84 mmol, 0.25 M solution in 15.3
mL tetrahydrofuran) at - 78 °C. After 1.5 h at this temperature, a solution of 2 (1.4 g, 7.13 mmol)
in tetrahydrofuran (14.2 mL) was added and the mixture was stirred at -78 °C overnight. After
completion, the mixture was quenched by adding 20% saturated ammonium chloride in 30%
ammonium hydroxide (1:4 sat. NH4Cl:NH4OH) and the mixture was stirred at rt for 1 h. The
mixture was then extracted with MTBE (x2) and the combined organic layers were dried over
sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The
crude material was purified by flash chromatography on silica gel (MTBE/hexane = 0/1 to 3/7) to
give 5b (1.53 g, 7.13 mmol, 66% yield) as a yellow oil, which solidified upon storage. LCMS m/z:
[M+Na]+ Calcd for C18H28O5Na 347.2; Found 347.2. Rf = 0.25-0.45 (20% EtOAC/hexane). 1H
NMR (CDCl3, 400 MHz): δH 4.91 (1H, d, J = 10.4 Hz), 4.43-4.30 (2H, m), 4.18 (2H, s), 3.72 (3H,
s), 3.18 (1H, d, J = 11.7 Hz), 2.87 (1H, q, J = 10.7 Hz), 2.54-2.49 (1H, m), 2.38-2.30 (1H, m),
2.00-1.89 (2H, m), 1.55-1.51 (2H, m), 1.38 (6H, s), 0.95 (3H, d, J = 7.0 Hz), 0.75 (3H, d, J = 6.9
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Note: Alternatively, the crude mixture (starting from 22 g of 2) can be filtered over short path
silica-gel and eluted with ethyl acetate until all colored residue has come out. The crude material
was again concentrated under reduced pressure, diluted with heptane (250 mL), stirred at -30 °C
for 30 minutes and filtered. The solid was washed with cold heptane (- 78 °C) to give 5b (9.84 g,
30.3 mmol, 27.1% yield) as a beige solid. Note: the keto form was major with only traces of the
enol form present.
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methyl
(1SR,2SR,3RS)-2-((2,2-dimethyl-1,3-dioxan-5-ylidene)methyl)-3-isopropyl-6-
methylenecyclohexane-1-carboxylate (9)
A mixture of 5b (1.29 g, 3.97 mmol) and methylidenetriphenylphosphane13 (1.09 g, 3.97
mmol) in tetrahydrofuran (26 mL) was heated at 60 °C for 24 h. After completion, the mixture was
concentrated under reduced pressure to give the crude product. Purification by flash
chromatography on silica gel (EtOAc/hexane = 0/1 to 15/85) gave 9 (1.05 g, 3.25 mmol, 82.0%
yield) as a white solid. HRMS (ESI) m/z: [M+Na]+ Calcd for C19H30O4Na 345.2042; Found
345.2032. Rf = 0.5 (20% EtOAc/hexane). IR (Nujol) νmax: 2992, 2954, 2845, 1729, 1646, 1434,
1379, 1355, 1261, 1238, 1216, 1189, 1150, 1067, 1043, 1015, 969, 910, 830, 728, 651, 513 cm–1.
1H NMR (CDCl3, 400 MHz): δH 4.83 (1H, d, J = 10.6 Hz), 4.77 (1H, s), 4.46-4.40 (2H, m), 4.34-
4.27 (1H, m), 4.13 (2H, dd, J = 18.1, 14.1 Hz), 3.65 (3H, s), 2.82 (1H, d, J = 11.0 Hz), 2.44-2.35
(2H, m), 1.99-1.93 (1H, m), 1.83-1.70 (2H, m), 1.37-1.34 (6H, m), 1.22-1.07 (2H, m), 0.85 (3H,
d, J = 6.9 Hz), 0.67 (3H, d, J = 6.9 Hz). Minor peaks corresponding to a diastereomer of 9 were
also observed (12:1 ratio favoring 9). 13C NMR (CDCl3, 100 MHz): δC 173.1, 145.0, 135.2, 124.7,
108.0, 99.1, 64.5, 60.2, 56.2, 51.5, 46.8, 42.7, 35.3, 28.1, 25.1, 24.4, 23.8, 21.6, 15.9. Minor peaks
corresponding to a diastereomer of 9 were also observed.
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Note: Unreacted starting material 5b could be recovered (220 mg, 0.678 mmol, 17.0 %) from the
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(1SR,2SR,3RS)-2-(3-hydroxy-2-(hydroxymethyl)prop-1-en-1-yl)-3-isopropyl-6-
methylenecyclohexane-1-carboxylic acid (11)5
3 M aqueous hydrochloric acid (5.2 mL, 15.5 mmol) was added to a solution of crude 9
(500 mg, 1.55 mmol) in methanol (1.0 mL) and the mixture was stirred at rt. The starting material
oiled out at the beginning, then slowly precipitated over 30-45 minutes. The mixture was stirred
until TLC indicated completion (1 h). The reaction mixture was cooled to 0 °C, then 2.5 M aqueous
sodium hydroxide (20 mL, 50 mmol) was added dropwise and the reaction mixture was allowed
to stir vigorously at rt. After overnight, solid material formed around the stir bar and was broken
down by sonication. The mixture was vigorously stirred at rt over 2 more days. After completion
(checked by LCMS), the mixture was acidified with 6 N aqueous HCl to pH = 3 at 0 °C, then the
solid was filtered via Buchner funnel and washed with water (x3). The collected solid was
triturated in tert-butyl methyl ether to give 11 (380 mg, 1.41 mmol, 92% yield) as a beige solid.
LCMS m/z: [M-H]- Calcd for C15H24O4 267.2; Found 267.1. 1H NMR (CDCl3, 400 MHz): δH 5.25
(1H, d, J = 10.7 Hz), 4.83 (1H, s), 4.61 (1H, s), 4.26-4.06 (4H, m), 2.91 (1H, d, J = 10.9 Hz), 2.65
(1H, q, J = 10.7 Hz), 2.48-2.43 (1H, m), 2.12-2.03 (1H, m), 1.93-1.81 (2H, m), 1.37-1.31 (1H, m),
1.21-1.10 (1H, m), 0.91 (3H, d, J = 7.0 Hz), 0.73 (3H, d, J = 6.9 Hz).
(5aSR,6RS,9aSR)-4-(hydroxymethyl)-6-isopropyl-9-methylene-5a,6,7,8,9,9a-
hexahydrobenzo[c]oxepin-1(3H)-one (12)5
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To a round bottom flask containing a solution of 11 (1.20 g, 4.60 mol, 1.00 equiv) in
dichloromethane (140 mL) was added triethylamine (1.9 mL, 14.0 mol, 3.00 equiv) and
dimethylaminopyridine (DMAP) (28.0 mg, 0.23 mmol, 0.05 equiv). The resulting solution was
cooled to 0 °C and bis(2-oxooxazolidin-3-yl)phosphinic chloride (BOP-Cl) (1.20 g, 4.90 mol, 1.05
equiv) was added. The mixture was warmed to 25 °C and stirred for 16 h. After completion, the
mixture was adjusted to pH = 2 with 0.5 M aqueous HCl solution and was extracted with
dichloromethane (x2). The combined organic layers were washed with saturated aqueous Na2CO3
and brine, dried over MgSO4, filtered and concentrated under reduced pressure. Acetone (100 mL)
was added (crystalline solid precipitates), the solid was filtered and the filtrate was concentrated
under vacuum to yield crude 12 as a green oil, which was used directly in the next step without
purification. LCMS m/z: [M+H]+ Calcd for C15H22O3 251.2; Found 251.1. Rf = 0.8 (100% EtOAc).
1H NMR (CDCl3, 400 MHz): δH 5.92 (1H, d, J = 3.8 Hz), 5.26 (1H, s), 4.97-4.93 (2H, m), 4.48
(1H, d, J = 14.5 Hz), 4.12-4.02 (2H, m), 3.65 (1H, d, J = 12.0 Hz), 2.42-2.37 (1H, m), 2.34-2.27
(1H, m), 2.07-1.98 (2H, m), 1.75-1.69 (1H, m), 1.41-1.34 (1H, m), 1.25-1.15 (2H, m), 0.88 (3H,
d, J = 6.9 Hz), 0.72 (3H, d, J = 6.9 Hz).
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(5aSR,6RS,9aSR)-6-isopropyl-9-methylene-1-oxo-1,3,5a,6,7,8,9,9a-octahydrobenzo[c]oxepine-
4-carbaldehyde (13)5
To a round bottom flask containing a solution of crude 12 (1.20 g, 4.60 mmol) in
dichloromethane (33.0 mL) was added manganese dioxide (4.20 g, 46.0 mmol, 10 equiv). The
resulting heterogenous mixture was heated to reflux for 16 h. The mixture was filtered on a short
silica plug and eluted with EtOAc. The resulting yellow solution was concentrated under reduced
pressure to give 13 as a pale yellow oil (0.750 g, 3.02 mmol, 66.0% yield over 2 steps). LCMS
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m/z: [M+H]+ Calcd for C15H20O3 249.1; Found 249.1. Rf = 0.2 (20% EtOAc/hexane). H NMR
(CDCl3, 400 MHz): δH 9.42 (1H, d, J = 0.5 Hz), 6.95-6.94 (1H, m), 5.45-5.44 (1H, m), 5.14-5.08
(2H, m), 4.97 (1H, dt, J = 15.1, 2.1 Hz), 3.83 (1H, d, J = 12.0 Hz), 2.65 (1H, tt, J = 11.4, 2.8 Hz),
2.50 (1H, dt, J = 13.7, 4.3 Hz), 2.18-2.08 (2H, m), 1.87-1.80 (1H, m), 1.61-1.54 (1H, m), 1.36
(1H, qd, J = 12.3, 4.5 Hz), 1.00 (3H, d, J = 6.8 Hz), 0.89 (3H, d, J = 6.9 Hz).
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(5aSR,6RS,9SR,9aSR)-6-isopropyl-1-oxo-1,5a,6,7,8,9a-hexahydro-3H-spiro[benzo[c]oxepine-
9,2'-oxirane]-4-carbaldehyde (14)5
To a reactor containing molybdenum trioxide9 (52 mg, 0.36 mmol, 0.20 equiv), 30%
aqueous hydrogen peroxide (74 µL, 0.72 mmol, 0.40 equiv) was added and the suspension was
heated to 40 °C and stirred for 2 h. The mixture was concentrated under reduced pressure to yield
a bright yellow solid. A solution of 13 (450 mg, 1.81 mmol, 1.0 equiv) in toluene (18.0 mL) was
added, followed by tert-butyl hydroperoxide (5.5 M solution in decane, 0.99 mL, 5.43 mmol, 3.0
equiv). The mixture was heated to 80 °C and stirred for 5 h. The mixture was cooled to 25 °C and
dimethyl sulfide (0.53 mL, 7.24 mmol, 4.0 equiv) was added. The mixture was stirred at 25 °C for
30 minutes. The suspension was filtered on a short silica plug and eluted with EtOAc. The resulting
yellow solution was concentrated under reduced pressure to give 14 as a pale yellow solid (310
mg, 1.17 mmol, 65% yield). LCMS m/z: [M+H]+ Calcd for C15H20O4 265.1; Found 265.2. Rf =
1
0.1 (20% EtOAc/hexane). H NMR (CDCl3, 400 MHz): δH 9.41 (1H, d, J = 0.4 Hz), 6.94-6.92
(1H, m), 5.12 (1H, d, J = 15.3 Hz), 4.91 (1H, dt, J = 15.3, 2.3 Hz), 3.82 (1H, dd, J = 5.1, 1.5 Hz),
3.55 (1H, d, J = 12.1 Hz), 2.77-2.70 (1H, m), 2.62 (1H, d, J = 5.1 Hz), 2.23-2.15 (1H, m), 1.98-
1.91 (1H, m), 1.86-1.78 (1H, m), 1.67-1.59 (2H, m), 1.53-1.42 (1H, m), 1.03 (3H, d, J = 6.9 Hz),
0.94 (3H, d, J = 6.9 Hz).
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To a solution of 14 (310 mg, 1.17 mmol) in tert-butanol (11.7 mL) and 2-methyl-2-butene
(9.00 mL) at 0 °C was added a solution of sodium chlorite (1.05 g, 11.7 mmol, 10.0 equiv) and
sodium dihydrogen phosphate (1.39 g, 11.7 mmol, 10.0 equiv) in water (4.25 mL). The resulting
solution was warmed to 25 °C and stirred for 2 h. After completion, sodium chloride (2.05 g, 35.1
mmol, 30.0 equiv) and acetic acid (1.00 mL) were added and the mixture was extracted with
EtOAc (x4) and dried over MgSO4. Heptane (20.0 mL) was gradually added during concentration
under vacuum, until the solution turned cloudy. The mixture was heated to boiling (crystalline
white solid precipitates). The suspension was filtered and the filter cake was washed with heptane
and dried under high vacuum to give rac-koningic acid as a white powder (0.285 g, 1.01 mmol,
87.0% yield). LCMS m/z: [M+H]+ Calcd for C15H20O5 281.1; Found 281.1. Rf = 0.8 (1%
AcOH/Et2O). 1H NMR (CD3OD, 400 MHz): δH 7.28 (1H, dd, J = 4.2, 1.9 Hz), 5.26 (1H, dt, J =
14.9, 2.0 Hz), 5.03 (1H, d, J = 14.9 Hz), 3.88 (1H, d, J = 12.1 Hz), 3.85 (1H, dd, J = 5.5, 1.6 Hz),
2.61 (1H, dddd, J = 12.1, 10.7, 4.2, 2.1 Hz), 2.54 (1H, d, J = 5.5 Hz), 2.14 (1H, pd, J = 6.9, 3.3
Hz), 1.96-1.90 (2H, m), 1.70-1.62 (1H, m), 1.52-1.39 (2H, m), 1.01 (3H, d, J = 6.9 Hz), 0.93 (3H,
d, J = 6.9 Hz).
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Supporting Information
Copies of 1H NMR ((iodomethyl)triphenylphosphonium iodide, 2,2-dimethyl-1,3-dioxan-5-one,
compounds 4, 5b, 9, 11, 12, 13, 14 and rac-koningic acid), 13C NMR (compounds 4 and 9), and
IR (compounds 4 and 9) spectra can be found in the supporting information.
Acknowledgements
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GB and BD thank Brian Albrecht, Jurre Kamphorst, Andy Long, Gad Soffer, Kelli Armstrong,
and Brooke Swalm for their feedback during the preparation of this manuscript. DC, BC, DG, EL,
and JM thank Guy Falardeau for useful suggestions.
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