Gram-Scale Synthesis of Tomatidine, a Steroid Alkaloid with Antibiotic Properties Against Persistent Forms of Staphylococcus aureus

Article abstract of DOI:10.1002/ejoc.202000051

We herein describe the first diastereoselective synthesis of the Solanum alkaloid tomatidine 1. The synthesis has been accomplished in 11 steps and 24.9 % overall yield (longest linear sequence). This methodology, which involves a convergent synthon insertion followed by a sequence of ring opening/nitrogen substitution/ring closing, allowed the generation of 1 on > 2 g scale. The synthetic challenge with the diastereoselective generation of the unusual spiroaminoketal moiety was solved through a combined azide reduction/addition sequence. The first diastereoselective synthesis of the phytosteroid yamogenin is also reported. Tomatidine has shown promising antibiotic properties against persistent forms of Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA). In particular, it possesses the unique ability to kill persistent forms of S. aureus and MRSA while simultaneously potentiating the antibiotic efficacy of aminoglycoside antibiotics against wild type strains of the bacteria.

Full text of DOI:10.1002/ejoc.202000051

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Gram-scale Synthesis of Tomatidine, a Steroid Alkaloid with
Antibiotic Properties Against Persistent Forms of Staphylococcus
aureus
Authors: Chad Normandin, François Malouin, and Eric Marsault
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202000051
Link to VoR: https://doi.org/10.1002/ejoc.202000051
01/2020

10.1002/ejoc.202000051
European Journal of Organic Chemistry
FULL PAPER
Gram-scale Synthesis of Tomatidine, a Steroid Alkaloid with
Antibiotic Properties Against Persistent Forms of
Staphylococcus aureus
Chad Normandin[a], Prof. Dr. François Malouin[b], and Prof. Dr. Eric Marsault*[a]
[a]
[b]
Institut de Pharmacologie de Sherbrooke
Université de Sherbrooke
3001, 12th Avenue N, Sherbrooke, Quebec, Canada J1H 5N4
E-mail: eric.marsault@usherbrooke.ca
Département de Biologie
Université de Sherbrooke
2500 Boul. de l’Université, Sherbrooke, Québec, Canada J1K 2X9
biofilm production all contribute to protect SCV from antibiotic
action, forming a reservoir that leads to persistence.2,4 SCV can
revert to the virulent WT upon cessation of treatment, leading to
infection relapses.5 Our team reported that 1 possesses potent
inhibitory activities against the SCV of S. aureus and MRSA, with
minimal inhibitory concentrations (MIC) as low of 0.06 µg/mL.6 We
further demonstrated that 1 targets the ATP synthase of S. aureus,
Abstract:
We herein describe the first diastereoselective synthesis of the
Solanum alkaloid tomatidine 1. The synthesis has been accomplished
in 11 steps and 24.9 % overall yield (longest linear sequence). This
methodology, which involves a convergent synthon insertion followed
by a sequence of ring opening/nitrogen substitution/ring closing,
allowed the generation of 1 on > 2g scale. The synthetic challenge
with the diastereoselective generation of the unusual spiroaminoketal
moiety was solved through a combined azide reduction/addition
sequence. The first diastereoselective synthesis of the phytosteroid
yamogenin is also reported. Tomatidine has shown promising
antibiotic properties against persistent forms of Staphylococcus
aureus (S. aureus) and methicillin-resistant S. aureus (MRSA). In
particular, it possesses the unique ability to kill persistent forms of S.
aureus and MRSA while simultaneously potentiating the antibiotic
efficacy of aminoglycoside antibiotics against wild type strains of the
bacteria.
a
protein important in energy production.7 We previously
elucidated the structure-activity relationship of 1 in order to
optimize its inhibition potential against SCV,7 and demonstrated
that 1 potentiates up to 16-fold the activity of aminoglycoside
antibiotics such as gentamicin or tobramycin against WT S.
aureus.8,9 These combined properties make tomatidine a target of
interest for use in combination with aminoglycosides to treat
simultaneously the WT and SCV forms of S. aureus and MRSA.
Introduction
In 2017, the World Health Organisation (WHO) identified S.
aureus, MRSA and other antibiotic-resistant bacterial species as
“high-priority” targets for the development of new antibiotics in the
fight against resistance.1 MRSA, resistant to β-lactams and
multiple other drugs, is widely found in hospital-acquired and
community-acquired infections.2 S. aureus and MRSA can adopt
two phenotypes, the prototypical wild-type (WT) and its small-
colony variants (SCV).3 The slow-growing SCV is the result of a
point mutation that occurs under pressure and affects the electron
transport chain. Slow growth, intracellular persistence and high
Figure 1. Tomatidine 1 and its glycosylated form tomatine 2
Tomatidine 1 is a steroid alkaloid found in more than 60 species
of the Solanum plant genus.10 It was first isolated and
characterized in 1948 as its naturally occurring C3-glycosylated
form tomatine 2 (Figure 1), which bears the Gal-Glu(Glu-Xyl)
tetrasaccharide.11 1 is biosynthesized from plant cholesterol via a
series of GLYCOALKALOID METABOLISM (GAME) enzymes.12
1
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10.1002/ejoc.202000051
European Journal of Organic Chemistry
FULL PAPER
Isolation from plants is currently the only means to access 1 and
2, both present in low concentrations in the leaves, roots and fruits
of Solanum plants. Tomatine 2 needs to be subjected to acidic
hydrolysis to deliver its aglycon form tomatidine 1. Tomatidine is
commercially available yet expensive, typically as a hydrochloride
salt of ca 85% purity. Structurally, it bears six rings (A-F, Figure
1), 12 stereogenic centers and a spiroaminoketal moiety (rings E-
F) also found in other biologically active products such as the
herbicide hydantocidin13, the phycotoxins azaspiracid A-E14,15 or
the immunosuppressant sanglifehrin A-D.16 The commercial price
of tomatidine (US$ 1,000-1,500/g) and its low availability limit
further optimization toward novel antibiotics, hence the strategic
need to develop a synthetic route. Recently, the synthesis of 25-
nortomatidine was reported by Czajkowska-Szczykowska et al. It
was obtained as a minor diastereoisomer in the synthesis of 25-
norsolasodine.17
Results and Discussion
Synthesis of iodide 10 (Scheme 2) was initiated by crotonoylation
of commercial (+)-camphorsultam using crotonyl chloride,
providing crystalline 6 in 96% yield. Subsequent alkylation using
LiHMDS and methyl iodide in THF/HMPA yielded 7 in 87% yield
and >50:1 d.r. (NMR).21,22 This methodology was preferred over
Evans‘ oxazolidinone approach, which is associated with lower
diastereoselectivity (d.r. < 3.6:1).23 7 was reductively cleaved
using LAH, and the resulting primary alcohol protected directly as
a tetrahydropyranyl (THP) ether to deliver alkene 8 in 93% yield
(2 steps). The enantiomeric excess of the alcohol was determined
to be >93% by chiral UPLC of the corresponding PMB ether (8a,
see supporting information). Hydroboration of alkene 8 using 9-
BBN followed by oxidative workup with sodium hydroxide and
hydrogen peroxide24 yielded primary alcohol 9 in 86% yield.
Alcohol 9 was transformed into the corresponding iodide 10 using
Appel conditions in 91% yield.25 The latter underwent iodide-
lithium exchange26 using t-butyllithium to generate organolithium
intermediate 11 in situ, to which was added 5 at -78°C to generate
hemiketal 12 in 82% yield (5 g scale). A total of 8.8 g of 12 was
prepared in this manner. To be noted, attempts to generate the
Grignard reagent derived from the corresponding bromide failed
to provide addition product 12 in useful yields.
Our goal was to develop the first synthesis of 1, aiming for a gram-
scale, efficient and cost-effective process from readily available
starting materials. Accordingly, the known dinorcholanic lactone 5
(Scheme 1) appeared as an attractive starting point. Indeed, 5
possesses the correct stereochemistry on rings A-D and C20
methyl. Following the methodology developed by Tian et al., Pd-
catalyzed hydrogenation, iodine-mediated Baeyer-Villiger
oxidation/ lactonisation of diosgenin 3 to 4 followed by TBDPS
protection of the C3 alcohol yielded dinorcholanic lactone 5 in 3
steps and high yields on 27g scale.18 Diosgenin, a triterpene
phytosteroid isolated industrially from Fenugreek,19,20 can be
purchased at low cost (US$ 0.30/g). Construction of rings E and
F, on the other hand, relied on the chiral synthesis of iodide 10.
R2 = H, Me
NHNs
OR2
O
(S)
(S)
N
H
H
O
H
H
H
Nitrogen
Substitution
Organometallic
Addition
H
H
TBDPSO
Oxonium
Condensation
H
HO
1
H
O
O
(R)
O
•Hydrogenation
•Oxidative cleavage
o
o
H
O
H
H
H
Tian et al.
80-83%
H
H
HO
R1O
I
(3)
Diosgenin
0.30 $ / g
Scheme 2. Synthesis of synthon 10 and hemiketal 12
H
(S)
OTHP
10
: R1 = H
: R1 = TBDPS
4
5
C5 synthon
27g prepared
With hemiketal 12 in hand, we set out to deprotect the THP group,
with the goal to convert it to an amine such as 14 (Scheme 3).27
However, even mild deprotection conditions (i.e. B10H14 (cat.) in
MeOH28, DDQ (cat.) in wet MeCN29, LiCl in wet DMSO30) failed to
yield alcohol 13 and repeatedly produced spiroketal 16 (Scheme
Scheme 1. Retrosynthetic analysis of 1
2
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10.1002/ejoc.202000051
European Journal of Organic Chemistry
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3). In more detail, when hemiketal 12 was subjected to pyridinium
p-toluenesulfonate (cat.) in methanol, ketal 15 was obtained
rapidly (< 2 min. at 0°C). Reaction was then brought to room
temperature for 1 h to yield spiroketal 16 after intramolecular
oxonium trapping of the newly generated alcohol. Further heating
yielded desilylated spiroketal 17. The structure of intermediate 16
(CCDC Deposition #1992541) was confirmed by single crystal X-
ray crystallography (Scheme 3), revealing a silylated and reduced
derivative of phytosteroid yamogenin 17, the 25S diastereomer of
diosgenin 3. This is not the first report of synthetic yamogenin31,
but it is the first reported diastereoselective synthesis. Yamogenin
possesses interesting anti-hyperlipidermic effects.32 Interestingly,
as opposed to tomatidine, its C25 methyl group prefers the axial
orientation, likely the result of anomeric stabilization by the two
spiro oxygen atoms. With spiroketal 16 in hand and the correct
stereochemistry installed on C25, we set out to introduce a
nitrogen source at the C26 position, before closing ring F to
generate the spiroaminoketal moiety. Various protocols have
been developed to open and functionalize the F ring of spiroketal
sapogenin steroids, often requiring strong oxidative and acidic
conditions33 or hazardous reagents.34 The Lewis acid-mediated
ring opening of steroidal spiroketals reported by Tian et al was
used.35 The TBDPS ether 16 being incompatible with these
conditions, it was removed by bringing the spiroketalisation step
to reflux in order to generate secondary alcohol 17 (Scheme 3) in
87% yield.36 The C3 alcohol was then protected as an acetate 18
in 94% yield (Scheme 4). The corresponding ω-halo enol ether
was then generated using BF3 etherate and lithium bromide,
followed by heating in DMF. The intermediate bromide was
substituted in situ by addition of sodium azide to the mixture to
afford ω-azido enol ether 19 in 96% yield. With the nitrogen group
installed on position C26, azide reduction to the amine and
subsequent cyclization onto the enol ether was achieved in one
step using trimethylsilyliodide (TMSI), generated by addition of
TMSCl to a solution of NaI in MeCN.35,37
Scheme 3. Synthesis of intermediate 17 and ORTEP diagram of intermediate 16 (50% Thermal Probability Ellipsoids)
in acidic conditions, with the neat result of in situ inversion of
configuration of the C25 methyl group.35 The subsequent
isomerized 25R amine intermediate yielded 21 as a minor product,
and 25S amine yielded tomatidine acetate 20 as the major
product. Spiroaminoketals 20 and 21 were finally subjected to
ester hydrolysis to afford 1 and 22 in 95% yield. Crystallization of
In these conditions, tomatidine acetate 20 was obtained in 67%
yield. Interestingly, its diastereoisomer 5,6-dihydrosolasodine
acetate 21 was also obtained in 11% yield. This result can be
explained by imine-enamine isomerisation of the reduced amine
3
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10.1002/ejoc.202000051
European Journal of Organic Chemistry
FULL PAPER
molecular sieves. Flash chromatography was performed on Silicycle
SiliaFlash P60 silica gel (230−400 mesh, 40−63 μm particle size).
Automated flash chromatography was performed using a Biotage Isolera
ISO-1SV automated flash chromatography system. Thin layer
chromatography (TLC) was performed on glass-backed Silicycle SiliaPlate
250 μm thickness, 60 Å porosity F-254 precoated plates from Silicycle.
Compounds were visualized with UV light (254 nm) then stained with
cerium ammonium molybdate followed by heating (steroids) or potassium
permanganate (KMnO4) followed by heating (non-steroids). Nuclear
magnetic resonance (NMR) spectra were obtained on a Bruker Ascend
400 spectrometer. Residual chloroform (CHCl3) signals were used as
internal reference for 1H (δ = 7.26 ppm) and 13C (δ = 77.1 ppm) spectra.
The following abbreviations were used to describe NMR multiplicities: br =
broad, s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sext
= sextuplet, sept = septuplet and m = multiplet. Coupling constants (J) are
given in Hertz (Hz). Infrared (IR) spectra were obtained on an ABB Bomem
MB104 spectrophotometer using a diamond-attenuated total reflectance
(ATR) accessory. All substances were directly applied on the ATR unit.
Frequency of absorption is given in cm−1. High-resolution mass spectra
synthetic 1 (CCDC Deposition # 1992540) and 22 (CCDC
Deposition # 1992542) from MeOH afforded crystals suitable for
x-ray diffraction, confirming their identity (Scheme 4). This
synthesis successfully delivered 2.35 g of synthetic Tomatidine.
(HRMS) were obtained on
a Nexera (Shimadzu) LC-QTOF mass
spectrometer coupled to maXis (Bruker) mass spectrometer (ESI) using
sodium formate as internal standard. Melting points were measured on an
Electrothermal MEL-TEMP 3.0 melting point apparatus. X-Ray diffraction
was performed on a KAPPA APEX-DUO (Bruker) diffractometer device
using a Cu radiation source.
Scheme 4. Completion of the synthesis and ORTEP diagram of Tomatidine 1
and 5,6-dihydrosolasodine 22 (50% Thermal Probability Ellipsoids)
2-(((S)-2-methylbut-3-en-1-yl)oxy)tetrahydro-2H-pyran (8). A solution
of 7 (28.3 g, 95.2 mmol, prepared as described by Cooksey, P. et al) in
Et2O (900 mL), was added dropwise over 2h to a suspension of lithium
aluminum hydride (7.22 g, 190 mmol, 2.0 eq.) in anhydrous Et2O (150 mL)
at 0°C. The resulting grey suspension was stirred vigorously and allowed
to warm to room temperature. When 7 was fully consumed as followed by
TLC (1 h), the mixture was slowly quenched by successive addition of
water (7.2 mL), 15% aqueous NaOH (7.2 mL) then water (21.6 mL). The
suspension was dried over sodium sulfate and stirred vigorously for 30 min.
The resulting suspension was then filtered through a glass-fritted funnel
and the residue washed repeatedly with Et2O (3x 100 mL) while manually
stirring the slurry in the funnel before filtering again. Filtrates were
combined and Et2O was removed via fractional distillation until minimal
Et2O remained (ca. 50 mL). A small aliquot of the reaction crude was put
aside for PMB derivatization and enantiomeric excess determination (see
supporting information). To this concentrated solution was added DCM
(250 mL), 3,4-Dihydro-2H-pyran (17.4 mL, 66.5 mmol, 2.0 eq.) and p-
toluenesulfonic acid monohydrate (362 mg, 1.90 mmol, 0.02 eq.). This
mixture was stirred until the alcohol was fully consumed as evidenced by
TLC (5 min). The mixture was then diluted with DCM (200 mL) then
washed with saturated aqueous NaHCO3 (40 mL). The organic layer was
dried with sodium sulfate then concentrated in vacuo. Purification by flash
chromatography (step gradient 0, 2.5, 5, 7.5, 10% EtOAc:Hex) yielded
tetrahydropyranyl ether 8 (15.1 g, 93%) as a light yellow oil. 1H NMR (400
MHz, CDCl3) δ (ppm) 5.85-5.75 (m, 1H); 5.05 (dd, 1H, J = 8.8 Hz, 1.2 Hz);
4.99 (dd, 1H, J = 5.2 Hz, 1.2 Hz); 4.57 (t, 1H, J = 3.6 Hz); 3.88-3.82 (m,
1H); 3.59 (ddd, 1H, J = 36.4 Hz, 9.6 Hz, 6.8 Hz); 3.51-3.46 (m, 1H); 2.46
(quint, 1H, J = 6.8 Hz); 1.86-1.77 (m, 1H); 1.74-1.65 (m, 1H); 1.61-1.48 (m,
Conclusion
In summary, we report the first synthesis of tomatidine 1 using an
11-step convergent sequence starting from cheap and
commercially available diosgenin 3. It delivered gram-scale
quantities with an overall yield of 24.9 % for the longest linear
sequence. The synthesis was achieved via the pivotal yamogenin
intermediate 17. This synthesis opens the way to larger amounts
of materials to further study the role and structure-activity
relationship of tomatidine 1 and analogs as potential antibiotic
agents against persistent forms of S. aureus and MRSA.
Experimental Section
General Information: All reactions were performed in flame-dried or oven-
dried glassware under an atmosphere of nitrogen using Teflon-coated stir
bars. Reactions ran above room temperature (23 °C) were heated in an oil
bath. Commercially available reagents were purchased from Sigma-
Aldrich with the highest available purity and used without purification.
Tetrahydrofuran (THF), methanol (MeOH), acetonitrile (MeCN), N,N-
dimethylformamide (DMF) were purchased as DriSolv® grade, stored and
used under argon atmosphere. Diethyl ether (Et2O) and pentane were
distilled over sodium under argon and kept dry using activated 3Å
4
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10.1002/ejoc.202000051
European Journal of Organic Chemistry
FULL PAPER
4H); 1.03 (dd, 3H, J = 6.8 Hz, 4.4 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm)
141.4, 114.1, 98.9, 94.8, 72.2, 63.0, 62.2, 37.9, 30.8, 25.6, 19.9, 19.6, 16.8.
FTIR-ATR (neat) ν (cm-1) 3036, 2943, 1652, 1635, 1124, 1074, 1035, 965,
820, 754. HRMS-ESI (m/z) calcd for C10H18O2: 193.1199 [M+Na]+ found :
193.1201 [M+Na]+.
eq.) previously dissolved in 13 mL anhydrous Et2O and 19 mL anhydrous
pentane at -78°C. This mixture was stirred for 25 min at -78°C until a
solution of 5 (5.02 g, 8.58 mmol) in pentane (25 mL) / anhydrous THF (20
mL) was transferred dropwise via canula. The mixture was stirred
vigorously at -78°C for 5 min, quenched with sat. aqueous NH4Cl (50 mL),
then warmed to room temperature. Water (75 mL) was added and the
(3S)-3-methyl-4-((tetrahydro-2H-pyran-2-yl)oxy)butan-1-ol (9). A 0.5M mixture was transferred to a separatory funnel. The aqueous layer was
solution of 9-BBN (47.6 mL, 23.8 mmol, 2.2 eq.) was added dropwise to a extracted with Et2O (3x 75 mL) and the combined organic phases were
cooled (0°C) solution of 8 (1.84 g, 10.8 mmol) dissolved in anhydrous THF dried over sodium sulfate and concentrated in vacuo. Purification by flash
(20 mL). The mixture was then slowly warmed to room temperature until 8 chromatography (0-30% EtOAc:Hex) afforded 12 (5.35 g, 82%) as a white
was consumed as evidenced by TLC (ca 1 h). The mixture was then cooled solid. 1H NMR (400 MHz, CDCl3) δ (ppm) 7.68-7.66 (m, 4H); 7.43-7.34 (m,
to 0°C before a premixed solution of 2M aqueous sodium hydroxide (16.2 6H); 4.58-4.53 (m, 2H); 3.87-3.83 (m, 1H); 3.61-3.48 (m, 3H); 3.21 (ddd,
mL, 32.4 mmol, 3 eq.) and aqueous 30% hydrogen peroxide (15.5 mL, 151 1H, J = 25.6 Hz, 9.6 Hz, 6.0 Hz); 1.05 (s, 9H); 1.01 (d, 3H, J = 6.8 Hz);
mmol, 14 eq.) was added dropwise. This mixture was then warmed to room 0.94 (dd, 3H, J = 6.8 Hz, 4.8 Hz); 0.80 (s, 3H) 0.75 (s, 3H). 13C NMR (100
temperature and stirred 1.5h before brine (100 mL) was added. The MHz, CDCl3) δ (ppm) 135.9, 135.1, 129.5, 127.5, 110.6, 98.9, 81.5, 77.4,
resulting mixture extracted with Et2O (3x 50 mL), combined organic fractions 72.9, 62.9, 62.3, 56.4, 54.4, 44.9, 41.0, 40.0, 38.5, 37.1, 36.5, 35.6, 35.2,
were dried on sodium sulfate, concentrated in vacuo and the product 33.6, 32.4, 31.8, 30.8, 28.7, 27.7, 27.1, 25.6, 21.1, 19.7, 19.3, 17.3, 16.5,
purified by flash chromatography (Step gradient, 0, 10, 20, 30, 35, 40, 45, 15.7, 12.5. FTIR-ATR (neat) ν (cm-1) 3421, 2929, 2855, 1451, 1380, 1110,
50% EtOAc:Hex) to yield alcohol 9 (1.74 g, 86%) as a colorless oil. 1H NMR 1062, 1028, 903, 863, 818, 740, 701, 612. HRMS-ESI (m/z) calcd for
(400 MHz, CDCl3) δ (ppm) 4.59-4.56 (m, 1H); 3.86-3.80 (m, 1H); 3.70 (sept, C48H72O5Si: 779.5041 [M+Na]+ found : 779.5046 [M+Na]+.
1H, J = 4.8 Hz); 3.66-3.55 (m, 2H); 3.52-3.47 (m, 1H); 3.28-3.18 (m, 1H);
2.49 (br s, 1H, OH); 1.90 (sept, 1H, J = 2.8 Hz); 1.83-1.75 (m, 1H); 1.72-
(4S,5'S,6aS,8aS,9S,10R,11aS)-5',6a,8a,9-
1.48 (m, 7H); 0.93 (dd, 3H, J = 6.8 Hz, 1.6 Hz). 13C NMR (100 MHz, CDCl3)
tetramethyldocosahydrospiro[naphtho[2',1':4,5]indeno[2,1-b]furan-
δ (ppm) 99.2, 99.0, 73.4, 73.2, 62.4, 61.2, 38.0, 31.3, 30.6, 25.5, 19.6, 17.7.
10,2'-pyran]-4-ol (17). p-Toluenesulfonic acid monohydrate (44.1 mg, 232
FTIR-ATR (neat) ν (cm-1) 3389, 2937, 2871, 1454, 1352, 1200, 1119, 1060,
μmol, 0.02 eq) was added to a stirring mixture of 12 (8.77 g, 11.6 mmol) in
1023, 974, 902, 867, 810. HRMS-ESI (m/z) calcd for C10H20O3: 211.1305
anhydrous MeOH (350 mL) at 0°C. After 2 min, hemiketal 12 (Rf 0.5, 25%
[M+Na]+ found : 211.1308 [M+Na]+.
EtOAc:Hex) was fully transformed into 15 (Rf 0.85) as evidenced by TLC.
The mixture was then warmed to room temperature. After 1 h, ketal 15 was
2-((S)-4-iodo-2-methylbutoxy)tetrahydro-2H-pyran (10). Iodine (1.19 g,
fully transformed into spiroketal 16 (Rf 0.90) by TLC. A crystal of 16
4.70 mmol, 1.3 eq.) was added in portions to a solution of PPh3 (1.14 g,
suitable for X-ray analysis could obtained by in vacuo concentration of an
4.34 mmol, 1.2 eq.) and imidazole (369 mg, 5.43 mmol, 1.5 eq.) in DCM (15
aliquot at this stage followed by purification by flash chromatography then
mL) previously cooled to 0°C. This mixture was stirred 20 minutes at 0°C
slow crystallization from MeOH. The mixture was then heated to 70°C.
before a solution of 9 (681 mg, 3.62 mmol) in DCM (10 mL) was added
After 3h, TLC showed complete conversion of intermediate 16 to 17 (Rf
dropwise over 10 minutes. The mixture was then warmed to RT and stirred
0.40). The mixture was then concentrated in vacuo and purified by flash
vigorously for 3h. It was then diluted with DCM (25 mL), quenched with a
chromatography (0-50% EtOAc:Hex) to yield 17 (4.19 g, 87%) as a white
10% sodium thiosulfate solution (10 mL) and the resulting aqueous layer
solid. 1H NMR (400 MHz, CDCl3) δ (ppm) 4.39 (q, 1H, J = 7.2 Hz); 3.94
was extracted with DCM (3x 30 mL). The combined organic layers were
(dd, 1H, J = 10.8 Hz, 2.4 Hz); 3.58 (sept, 1H, J = 4.8 Hz); 3.29 (d, 1H, J =
washed with brine (15 mL), dried on sodium sulfate and concentrated in
11.2 Hz); 1.07 (d, 3H, J = 7.2 Hz); 0.98 (d, 3H, J = 6.8 Hz); 0.81 (s, 3H);
vacuo. Purification by flash chromatography (0-5% EtOAc:Hex) afforded 10
0.75 (s, 3H). 13C NMR (100 MHz, CDCl3) δ (ppm) 109.7, 81.1, 71.4, 65.3,
(1.00 g, 91%) as a colorless oil. (NOTE: product is light-sensitive and needs
62.2, 56.4, 54.5, 44.9, 42.3, 40.7, 40.2, 38.3, 37.1, 35.7, 35.3, 32.4, 31.7,
to be stored in a cold, dark room under inert atmosphere). 1H NMR (400
31.6, 28.7, 27.2, 26.1, 25.9, 21.2, 16.6, 16.2, 14.5, 12.5. FTIR-ATR (neat)
MHz, CDCl3) δ (ppm) 4.55 (q, 1H, J = 3.6 Hz); 3.85-3.80 (m, 1H); 3.62-3.56
ν (cm-1) 3255, 2928, 2847, 1448, 1368, 1221, 1174, 1056, 983, 953, 920,
(m, 1H); 3.52-3.47 (m, 1H); 3.32-3.26 (m, 1H); 3.24-3.17 (m, 2H); 2.08-1.96
849, 774. HRMS-ESI (m/z) calcd for C27H44O3: 439.3183 [M+Na]+ found :
(m, 1H); 1.91-1.77 (m, 2H); 1.75-1.65 (m, 2H); 1.61-1.49 (m, 4H); 0.93 (dd,
439.3178 [M+Na]+.
3H, J = 6.8 Hz, 4.4 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm) 98.9, 72.0,
(4S,5'S,6aS,8aS,9S,10R,11aS)-
62.3, 38.2, 34.7, 30.8, 25.6, 19.6, 16.5, 5.1. FTIR-ATR (neat) ν (cm-1) 2938,
5',6a,8a,9tetramethyldocosahydrospiro[naphtho[2',1':4,5]indeno[2,1-
2869, 1453, 1351, 1183, 1119, 1063, 1031, 973, 903, 868, 814. HRMS-ESI
b]furan-10,2'-pyran]-4-yl acetate (18). Acetic anhydride (7.60 mL, 80.5
(m/z) calcd for C10H29IO2: 321.0322 [M+Na]+ found : 321.0328 [M+Na]+.
mmol, 8.0 eq.) was added to a stirring mixture of 17 (4.19 g, 10.1 mmol)
previously dissolved in pyridine (40 mL) at 50°C. This mixture was stirred
(4S,6aS,8aS,9S,10R,11aS)-4-((tert-butyldiphenylsilyl)oxy)-6a,8a,9-
at 50°C until 17 was fully consumed as shown by TLC (ca 2h), cooled to
trimethyl-10-((3S)-3-methyl-4-((tetrahydro-2H-pyran-2-
room temperature then poured into stirred ice-cold water (150 mL). The
yl)oxy)butyl)octadecahydro-1H-naphtho[2',1':4,5]indeno[2,1-b]furan-
resulting suspension was stirred vigorously for 15 min while cooled in an
10-ol (12). Freshly titrated t-BuLi38 (1.63 M in Pentane, 21.3 mL, 34.7 mmol,
ice bath then vacuum filtered using a Buchner funnel. The resulting solid
4.05 eq.) was added dropwise to a mixture of 10 (5.12 g, 17.2 mmol, 2.0
5
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10.1002/ejoc.202000051
European Journal of Organic Chemistry
FULL PAPER
was washed with ice-cold water and further dried by vacuum filtration 15
min before it was dried in vacuo to yield 18 (4.35 g, 94%) as a white solid.
1H NMR (400 MHz, CDCl3) δ (ppm) 4.67 (sept, 1H, J = 4.8 Hz); 4.39 (q,
1H, J = 7.2 Hz); 3.94 (dd, 1H, J = 10.8 Hz, 2.8 Hz); 3.29 (d, 1H, J = 11.2
Hz); 2.01 (s, 3H); 1.07 (d, 3H, J = 7.2 Hz); 0.98 (d, 3H, J = 6.8 Hz); 0.83
(s, 3H); 0.75 (s, 3H). 13C NMR (100 MHz, CDCl3) δ (ppm) 170.8, 109.8,
81.0, 73.8, 65.2, 62.1, 56.3, 54.3, 44.7, 42.2, 40.6, 40.1, 36.8, 35.7, 35.2,
34.1, 32.2, 31.8, 28.6, 27.6, 27.2, 26.0, 25.9, 21.6, 21.1, 16.6, 16.2, 14.4,
12.4. FTIR-ATR (neat) ν (cm-1) 2923, 2898, 2847, 1724, 1447, 1379, 1240,
1174, 1132, 1049, 1028, 987, 920, 854, 611. HRMS-ESI (m/z) calcd for
C29H46O4: 481.3288 [M+Na]+ found : 481.3277 [M+Na]+
δ (ppm) 4.67 (sept, 1H, J = 4.8 Hz); 2.76-2.68 (m, 2H); 2.01 (s, 3H); 0.95
(d, 3H, J = 6.8 Hz); 0.85 (d, 3H, J = 6.4 Hz); 0.83 (s, 3H); 0.81 (s, 3H). 13
C
NMR (100 MHz, CDCl3) δ (ppm) 170.8, 99.2, 78.6, 73.8, 62.1, 55.8, 54.4,
50.4, 44.8, 43.2, 41.0, 40.3, 36.8, 35.7, 35.2, 34.1, 32.8, 32.3, 31.2, 28.7,
28.6, 27.6, 26.8, 21.6, 21.2, 19.5, 17.1, 16.0, 12.4. FTIR-ATR (neat) ν (cm-
1) 2919, 2847, 1727, 1451, 1365, 1237, 1132, 1030, 974, 888, 743, 660.
HRMS-ESI (m/z) calcd for C29H47NO3: 458.3629 [M+H]+ found : 458.3630
[M+H]+.
Tomatidine (1) A 3M aqueous solution of NaOH (9.94 mL, 29.8 mmol, 5.0
eq.) was added to a solution of 20 (2.73 g, 5.96 mmol) previously dissolved
in DCM (25 mL) and MeOH (75 mL). The mixture was then stirred at room
temperature until 20 was fully consumed as shown by TLC (2h). pH was
then adjusted to 7-8 using 1N HCl (ca 12 mL) and the mixture was
concentrated in vacuo, suspended in water (50 mL) then extracted with
chloroform (3x 75 mL). The combined organic layers were dried on sodium
sulfate and concentrated in vacuo. Purification by flash chromatography
(10-60% EtOAc:Hex, 1% Et3N) afforded Tomatidine 1 (2.35 g, 95%) as a
white solid. A crystal suitable for X-ray analysis was obtained by slow
crystallization from MeOH. 1H NMR (400 MHz, CDCl3) δ (ppm) 4.12 (q, 1H,
8.4 Hz); 3.57 (sept, 1H, J = 4.8 Hz); 2.79-2.76 (m, 1H); 2.72 (t, 1H, J = 10.8
Hz); 2.01-1.95 (m, 1H); 0.95 (d, 3H, J = 7.2 Hz); 0.85 (d, 3H, J = 6.4 Hz);
0.81 (s, 6H). 13C NMR (100 MHz, CDCl3) δ (ppm) 99.2, 78.7, 71.4, 62.1,
55.9, 54.6, 50.4, 44.9, 43.2, 41.0, 40.4, 38.4, 37.1, 35.7, 35.2, 32.8, 32.5,
31.7, 31.2, 28.8, 28.7, 28.4, 26.8, 21.2, 19.5, 17.1, 16.0, 12.5. FTIR-ATR
(neat) ν (cm-1) 3330, 2916, 2852, 1445, 1382, 1137, 1050, 975, 900, 874,
787, 657, 628. HRMS-ESI (m/z) calcd for C27H45NO2: 416.3523 [M+H]+
found : 416.3520 [M+H]+.
(4S,6aS,8aS,11aS)-10-((S)-4-azido-3-methylbutyl)-6a,8a,9-trimethyl-
2,2a,3,4,5,6,6a,6b,7,8,8a,8b,11a,12,12a,12b-hexadecahydro-1H-
naphtho[2',1':4,5]indeno[2,1-b]furan-4-yl acetate (19). Dry LiBr (8.01 g,
92.2 mmol, 10.0 eq.) and 18 (4.23 g, 9.22 mmol) were loaded into a flame-
dried 500ml round bottom flask. DCM (80 mL) and anhydrous MeCN (40
mL) were then added and the mixture stirred vigorously before BF3•Et2O
(11.4 mL, 92.2 mmol, 10.0 eq.) was added dropwise. The reaction was
stirred vigorously at RT for 3h until two slightly more polar spots (Rf 0.4
(major) and 0.35 (minor)) appeared. The mixture was slowly quenched
with sat. aqueous NaHCO3 (CAUTION : Vigorous gas generation) until no
more bubbling occurred, diluted with water then extracted with DCM (3x
75 mL). The combined organic layers were washed with brine, dried on
sodium sulfate then concentrated in vacuo. The crude mixture was then
dissolved in DMF (40 mL) and stirred at 70°C for 2h before addition of
sodium azide (1.80 g, 27.7 mmol, 3.0 eq.). The mixture was stirred an
additional 2h at this temperature then cooled to room temperature, diluted
with water and extracted with DCM (3x 75 mL). The combined organic
phases were washed with brine (40 mL), dried on sodium sulfate then
concentrated in vacuo. Purification by flash chromatography (0-15%
EtOAc:Hex) yielded 19 (4.28 g, 96%) as a white waxy solid. 1H NMR (400
MHz, CDCl3) δ (ppm) 4.74-4.64 (m, 2H); 3.23 (dd, 1H, J = 12.0 Hz, 5.6
Hz); 3.09 (dd, 1H, J = 12.0 Hz, 7.2 Hz); 2.45 (d, 1H, J = 10.0 Hz); 2.18-
2.07 (m, 3H); 2.01 (s, 3H); 1.57 (s, 3H); 0.96 (d, 3H, J = 6.8 Hz); 0.83 (s,
3H); 0.65 (s, 3H). 13C NMR (100 MHz, CDCl3) δ (ppm) 170.5, 151.2, 103.9,
84.3, 73.5, 64.3, 57.6, 54.7, 54.2, 44.6, 43.5, 39.6, 36.7, 35.5, 34.9, 34.0,
33.0, 32.3, 31.4, 28.5, 27.4, 23.1, 21.4, 21.1, 17.4, 14.2, 12.2, 11.6. FTIR-
ATR (neat) ν (cm-1) 2915, 2845, 2095, 1731, 1449, 1367, 1237, 1029, 959,
896, 860, 664, 608. HRMS-ESI (m/z) calcd for C29H45N3O3: 506.3353
[M+Na]+ found : 506.3340 [M+Na]+.
5,6-Dihydrosolasodine (22)
A 3M aqueous solution of NaOH (401 μL, 1.20 mmol, 5.0 eq.) was added
to a solution of 21 (110 mg, 240 μmol) previously dissolved in DCM (1 mL)
and MeOH (3 mL). The mixture was then stirred at room temperature until
21 was fully consumed as shown by TLC (2h). pH was then adjusted to 7-
8 using 1N HCl (ca 1 mL) and the mixture was concentrated in vacuo,
suspended in water (5 mL) then extracted with chloroform (3x 10 mL). The
combined organic layers were dried on sodium sulfate and concentrated
in vacuo. Purification by flash chromatography (10-60% EtOAc:Hex, 1%
Et3N) afforded 5,6-Dihydrosolasodine 22 (99 mg, 95%) as a white solid. A
crystal suitable for X-ray analysis was obtained by slow crystallization from
MeOH. 1H NMR (400 MHz, CDCl3) δ (ppm) 4.28 (q, 1H, J = 7.6 Hz) 3.56
(sept, 1H, J = 4.8 Hz) 2.68-2.65 (m, 1H) 2.59 (t, 1H, J = 11.2 Hz) 2.02-1.95
(m, 1H) 1.88 (t, 1H, J = 7.2 Hz) 1.80-1.77 (m, 1H) 0.94 (d, 3H, J = 6.8 Hz)
0.83 (d, 3H, J = 6.0 Hz) 0.81 (s, 3H) 0.77 (s, 3H) 13C NMR (100 MHz,
CDCl3) δ (ppm) 98.3, 79.2, 71.3, 62.9, 56.4, 54.5, 47.6, 44.9, 41.4, 40.9,
40.3, 38.3, 37.1, 35.7, 35.2, 34.0, 32.4, 32.2, 31.6, 31.2, 30.2, 28.7, 21.2,
19.4, 16.7, 15.4, 12.5 FTIR-ATR (neat) ν (cm-1). 3358, 2930, 2846, 1673,
1447, 1347, 1129, 1065, 1047, 974, 957, 884, 774, 680. 670. HRMS-ESI
(m/z) calcd for C27H45NO2: 416.3523 [M+H]+ found : 416.3527 [M+H]+.
Tomatidine acetate (20). Sodium iodide (2.93 g, 19.6 mmol, 2.2 eq) and
19 (4.30 g, 8.89 mmol) were dissolved in anhydrous MeCN (120 mL). This
mixture was stirred for 30 min before a solution of freshly distilled
trimethylsilyl chloride (2.60 mL, 20.4 mmol, 2.3 eq) in MeCN (20 mL) was
added dropwise via canula. The resulting mixture was stirred for 30 min at
room temperature, quenched with saturated aqueous sodium thiosulfate
(20 mL), pH ajusted to 9-10 using 5% NaOH (8 mL) then concentrated
under reduced pressure. The remaining suspension was extracted with
chloroform (3x 75 mL). The combined organic layers were washed with
brine, dried with sodium sulfate, then concentrated in vacuo. Purification
by flash chromatography (0-40% EtOAc:Hex, 1% Et3N) afforded 20 (2.73
g, 67%) and 21 (440 mg, 11%) as white solids. 1H NMR (400 MHz, CDCl3)
6
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10.1002/ejoc.202000051
European Journal of Organic Chemistry
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8
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9
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