Convergent Synthesis of Menaquinone-7 (MK-7)

Article abstract of DOI:10.1021/acs.oprd.6b00037

A practical synthesis of menaquinone-7 (MK-7, Vitamin K₂) in the all-trans form was designed. Stereoselective synthesis of MK-7 was achieved through a "1 + 6" convergent strategy by condensation of two building blocks, menadione monoprenyl derivative (fragment "1") with hexaprenyl bromide (fragment "6", 82%). Pd-catalyzed desulfonation with LiEt₃BH (78%) was followed by oxidation of the hydroquinone moiety using ammonium cerium(IV) nitrate (72%). The major challenge in our methodology was the preparation of all-trans hexaprenyl bromide by coupling of two triprenyl units derived from trans, trans-farnesol. Manufacturing on a pilot scale was accomplished through our approach. The scalable method was designed especially for a large, kilogram-scale production from easily available intermediates. Furthermore, the proposed methodology avoids many chromatographic purifications and allows for a relatively cost-effective manufacturing. Moreover, our synthesis yielded high-purity (99.9%) final product MK-7, which can be used as a dietary supplement as well as an active pharmaceutical ingredient.

Full text of DOI:10.1021/acs.oprd.6b00037

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Full Paper
Convergent synthesis of menaquinone-7 (MK-7)
Aneta Baj, Piotr Wa#ejko, Andrzej Kutner, #ukasz
Kaczmarek, Jacek Witold Morzycki, and Stanislaw Witkowski
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00037 • Publication Date (Web): 06 May 2016
Downloaded from http://pubs.acs.org on May 8, 2016
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CONVERGENT SYNTHESIS OF
MENAQUINONEꢀ7 (MKꢀ7)
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Aneta Baj,† Piotr Wałejko,† Andrzej Kutner,‡ Łukasz Kaczmarek, ‡ Jacek W. Morzycki, †
Stanisław Witkowski*,†
†Institute of Chemistry, University of Białystok, Ciołkowskiego 1K, 15ꢀ245 Białystok, Poland
‡Pharmaceutical Research Institute, Rydygiera 8, 01ꢀ793 Warszawa, Poland
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GRAPHICAL ABSTRACT
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ABSTRACT:
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A practical synthesis of menaquinoneꢀ7 (MKꢀ7, vitamin K2) in the allꢀtrans form was
designed. Stereoselective synthesis of MKꢀ7 was achieved through a “1 + 6” convergent strategy
by condensation of two building blocks, menadione monoprenyl derivative (fragment “1”) with
hexaprenyl bromide (fragment “6”, 82%). Pdꢀcatalyzed desulfonation with LiEt3BH (78%) was
followed by oxidation of the hydroquinone moiety using ammonium cerium(IV) nitrate (72%).
The major challenge in our methodology was the preparation of allꢀtrans hexaprenyl bromide by
coupling of two triprenyl units derived from trans,transꢀfarnesol. Manufacturing on a pilot scale
was accomplished through our approach. The scalable method was designed especially for
a large, kgꢀscale production from easily available intermediates. Furthermore, the proposed
methodology avoids many chromatographic purifications and allows for a relatively costꢀ
effective manufacturing. Moreover, our synthesis yielded highꢀpurity (99.9%) final product MKꢀ
7, which can be used as a dietary supplement as well as an active pharmaceutical ingredient.
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Keywords: vitamin K2, menaquinoneꢀ7, prenylation, convergent synthesis, MKꢀ7
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INTRODUCTION
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Vitamin K is the family of fatꢀsoluble compounds that contain a 2ꢀmethylꢀ1,4ꢀnaphthoquinone
moiety and differ in an alkenyl substituent at the 3ꢀposition.1 There are two natural forms of
vitamin K: vitamin K1 (phylloquinone or phytonadione) bearing a long phytyl side chain and
vitamin K2 (menaquinones) with a polyprenyl side chain (Figure 1). Vitamin K3 (menadione) is
a synthetic form of vitamin K which acts as a provitamin. However, it is not recommended for
humans as a dietary supplement. According to some reports, menadione might be toxic to the
liver, and in addition may lead to hemolytic anemia and allergic reactions.2–4
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Figure 1. Chemical structure of vitamins K.
The major dietary source of vitamin K is phylloquinone, which is synthesized by plants and
algae.2 High concentration of vitamin K1 is present in green leafy vegetables (e.g. broccoli,
iceberg lettuce, spinach) and some plant oils (e.g. soybean oil, olive oil).5 Vitamin K2 is a group
of related compounds described as MKꢀn, where n is the number of unsaturated isoprenoid units
in the side chain (from 4 to 13)6. The chain length and number of double bonds have an
influence on the activity and bioavailability. The main dietary source of vitamin K2 are animal
products e.g. eggs, meet and fermented foods such as natto, cheese, curd and sauerkraut.2,7,8
High concentration of MKꢀ7 (Figure 2) is present in traditional Japanese “natto” (900ꢀ1200
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ꢁg/100 g9, when the content of vitamin K1 in spinach is 380 ꢁg/100 g5). Menaquinones are
synthetized by bacteria, except for MKꢀ4 (menatetrenone),4 which can be formed in direct
conversion of dietary phylloquinone or by alkenylation of menadione.10 In humans, deficiency of
vitamin K2 may occur after prolonged treatment with sulfonamide antibacterials when bacterial
flora may be partially or completely destroyed.
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Figure 2. Chemical structure of Menaquinone-7 (MK-7).
It is commonly known that vitamin K is required for proper blood clotting.8 In the liver it
catalyzes the synthesis of prothrombin, the inactive precursor of thrombin, which converts
fibrinogen of blood plasma into fibrin. Vitamin K is an essential cofactor in the γꢀglutamyl
carboxylation pathway. γꢀGlutamyl carboxylase is responsible for postꢀtranslational conversion
of some glutamate residues to γꢀcarboxyglutamic acid residues in osteocalcin.2,11
Recent studies have shown that vitamin K play an important role in bone health,4,9,12,13
reduction of vascular calcification and cardiovascular risk processes.7,14 Some data suggest that
vitamins D and K work synergistically on bone density.9 Furthermore, vitamin K reveals
antibacterial,15,16 antiꢀinflammatory and anticancer activity.2 For example, vitamin K2 has been
reported to induce apoptosis in hepatocellular carcinoma and leukemia.17–19
Currently, vitamin K1 (synthetic and natural), MKꢀ4 (synthetic), and MKꢀ7 (from natto) are
used in food supplementation. With regard to therapeutic aspects, menaquinoneꢀ4 (MKꢀ4) and
menaquinoneꢀ7 (MKꢀ7) are the most important forms of vitamin K2. Nowadays, there is a
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growing interest in MKꢀ7 supplementation. Vitamin K1 is the primary dietary form of vitamin K
but is poorly absorbed by the human body (5ꢀ15%). Vitamin MKꢀ4 has a poor bioavailability at
nutrition level dose (420 ꢁg/day), requires high doses (15ꢀ45 mg/day) and frequent
administration. On the contrary, vitamin MKꢀ7 is more lipophilic, has better bioavailability in
small intestine and longer halfꢀlife (about 3 days) compared to vitamin MKꢀ4 (1 h) and
phylloquinone (several hours). Moreover, MKꢀ7 is highly effective in the carboxylation of
osteocalcin (OC) at nutritional dose (45ꢀ90 ꢁg/day), where larger amount of MKꢀ4 (1500
ꢁg/day) is required to activate OC. 4,20–22
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All natural menaquinones have allꢀtrans configurations for the appropriate side chain double
bonds. In view of this was planned to design an efficient and stereoselective method for
preparing high purity allꢀtrans vitamin MKꢀ7 which would meet the quality requirements
approved for the dietary supplements.
RESULTS AND DISCUSSION
Vitamin MKꢀ7 (1) has seven all-trans double bonds in the side chain. Based on retrosynthetic
analysis vitamin MKꢀ7 can be obtained by coupling “1 + 6” (Scheme 1). Key to this strategy is a
reaction of αꢀphenylsulfonyl carbanion generated in situ from protected menadiol derivative 2
with allꢀtransꢀhexaprenyl bromide (3). The methodology based on formation of a phenylsulfonyl
derivative followed by its reaction with an electrophile, such as halide, in the presence of base
(e.g. potassium tertꢀbutoxide) is widely used in syntheses of many important natural products
such as solanesol23 or ubiquinone (coenzyme Q10)24–26
.
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Compound 2 is a convenient substrate due to the presence of a monoprenyl chain, which can
be extended by further prenyl units. Additionally, the presence of sulfonyl group may facilitate
purification of this product by crystallization.
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Scheme 1. Retrosynthetic analysis of Menaquinone-7 (MK-7).
The required hexaprenyl fragment 3 can be prepared from commercially available
terpenes, e.g. geraniol, farnesol or geranylgeraniol according to one of the three strategies:
“2+2+2”, “2+4” or “3+3” (Scheme 1). Preliminary experiments allowed to choose “3+3”
strategy and inexpensive (E,E)ꢀfarnesol as a convenient substrate. In this case, it was planned to
synthesize hexaprenyl moiety via coupling of two building blocks containing three prenyl units
using sulfonyl methodology. This strategy can be performed in two variants, depending on the
structure of the substrate as depicted in Scheme 2. Allyl halide can be reacted with polyprenyl
sulfone to form sulfonated polyprenol derivative. Sulfonyl coupling group was chosen due to its
easy preparation and removal by selective reduction.27
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Scheme 2. Synthetic route to bromide 3.
Vitamin MKꢀ7 (1) was prepared from the readily available and inexpensive starting
materials as shown in Scheme 3. Compound 2 was obtained in moderate yield (24% from
menadione) in a simple and efficient way according to slightly modified procedures.26,28–31 The
formation of intermediate 4 involved twoꢀstep protocol including reduction of menadione (S1)
with sodium dithionate (Na2S2O4) followed by methylation (Me2SO4). In the parallel
experiment, the reaction of isoprene (S2) with benzenesulfonyl chloride in the presence of
copper chloride and triethylamine hydrochloride as catalysts afforded a mixture of E/Z-isomers.
The desired compound 5 (Eꢀisomer) was isolated by crystallization of the crude product from 2ꢀ
propanol. Finally, FriedelꢀCrafts alkenylation of 4 by chlorosulfone 5 provided the desired
product 2. After purification by DFC, followed by crystallization from methanol the HPLC
purity of sulfone 2 (E-isomer, exclusively) achieved 99.6%.
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Scheme 3. Synthesis of Menaquinone-7 (MK-7).a
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aReagents and conditions: a) 1. Na2S2O4, AcOEt/water, RT; 2. Me2SO4, K2CO3, acetone,
reflux; b) PhSO2Cl, CuCl, Et3N·HCl, acetonitrile, 60 °C; c) SnCl4, 1,2ꢀdichloroethane, 80 °C; d)
Ac2O, Py, RT; e) 1. SeO2, tꢀBuO2H, salicylic acid, CH2Cl2, RT; 2. NaBH4, THF/MeOH, ꢀ10 °C;
f) 1. PBr3, THF, 0 °C; 2. PhSO2Na, DMF, RT; g) 1. tꢀBuOK, THF/DMF, ꢀ78 °C; 2. trans, transꢀ
farnesyl bromide; 3. NaOH, MeOH, RT; h) LiEt3BH, Pd(dppe)Cl2, THF, 0 °C to RT; i) PBr3,
THF, 0 °C; j) tꢀBuOK, THF/DMF, ꢀ20 °C; k) CAN, CH2Cl2/acetonitrile /water, 0 °C.
The current method requires functionalization at the head of the isoprenoid skeleton. For
this purpose (E,E)ꢀfarnesol (S3) was acetylated and then subjected to stereoselective allylic
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oxidation32,33 at the terminal methyl group. The oxidation using 10 mol% of SeO2 and 3.6 equiv.
of tꢀBuO2H in the presence of 10 mol% of salicylic acid followed by reduction with NaBH4 gave
allylic alcohol 7 in 29% yield. In the next step, hydroxyl in 7 was replaced by bromine with PBr3
and the freshly prepared bromide, without purification, was converted to the sulfone 8. The
reaction of sulfurꢀstabilized allylic carbanion, generated from 8 by nꢀBuLi in THF/DMF, with
(E,E)ꢀfarnesyl bromide followed by deacetylation afforded compound 9. Desulfonation of 9 was
carried out using lithium triethylborohydride (Superꢀhydride, LiEt3BH) and Pd(dppe)Cl2 (dppe =
1,2ꢀbis(diphenylphosphino)ethane) as a catalyst in anhydrous THF (76% yield). The resulting
hexaprenol (10) was characterized by 1H and 13C NMR. The carbon chemical shift for one of the
geminal vinylic methyl groups amounts 25.6 ppm, while for other methyl groups appear in the
range of 15ꢀ18 ppm. These data confirm that all the double bonds are of trans geometry. The
purity of all-trans hexaprenol 10 was determined by HPLC (91%).
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Hexaprenol 10 was reacted with PBr3 and the resulting bromide was used directly for
alkenylation. Coupling of menadione derivative 2 with hexaprenyl bromide 3 produced the
required carbon skeleton of menaquinone MKꢀ7 in 82% yield. Finally, desulfonation (LiEt3BH,
Pd(dppe)Cl2) followed by oxidation (cerium(IV) ammonium nitrate, CAN) gave the target
product 1 as a yellow oil. After purification by DFC, followed by crystallization from ethanol
and ethyl acetate vitamin MKꢀ7 (1) was obtained as a yellow powder (99.9% purity by HPLC).
A different approach to the synthesis of allꢀtransꢀhexaprenol (10) was also examined (Scheme
4). Alcohol 7 was transformed into bromide 7a, which reacted in the same manner as in the first
approach, with phenylsulfone 8a obtained from trans,transꢀfarnesol. Deacetylation of crude
coupling product afforded alcohol 9a. No isomeric products of the coupling at the γ position was
observed. Reductive removal of the sulfonyl group afforded hexaprenol 10 in high yield (73%).
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The NMR spectra were identical with those disclosed earlier and thus confirmed the molecular
structure of the product.
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Scheme 4. Alternative approach to the synthesis of all-trans hexaprenol (10).
CONCLUSIONS
In conclusion, we have developed an efficient synthetic pathway for preparation of vitamin K2
in MKꢀ7 form starting from easily available substrates. Our “1 + 6” convergent strategy is based
on condensation of monoprenyl derivative of menadione (2; fragment “1”) with hexaprenyl
bromide (3; fragment “6”). Compound 2 was prepared in a moderate yield (24% from
menadione) in a simple and efficient way using crystallization as a method for the isolation of
highly pure desired Eꢀisomer. In turn, bromide 3 was obtained from readily available (E,E)ꢀ
farnesol in six steps in 7% overall yield. The main advantage of the reported method is high
purity of both substrates. The final product was obtained from 2 and 3 in three steps in 46%
overall yield. The proposed method allows to isolate menaquinoneꢀ7 in the all-trans
configuration with high purity (99.9%) in moderate overall yield (11% starting from
menadione). This procedure can be used on industrial scale. Furthermore, very high purity of the
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final product (MKꢀ7) allows to use it as a dietary supplement. The described methodology in a
large production scale has been patented.34
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EXPERIMENTAL SECTION
General. Starting materials and reagents were obtained from commercial sources and used
without further purification. Solvents were dried by distillation over appropriate drying agents
under argon atmosphere. All moisture and airꢀsensitive reactions were carried out under argon
using ovenꢀdried glassware. Thin layer chromatography (TLC) was performed using Merck
silica gel plates (0.25 mm, 60Fꢀ254), visualized by spraying with H2SO4/MeOH solution (1:9,
v/v) followed by heating. Flash chromatography (FC) and dryꢀcolumn flash chromatography
(DFC) were performed on J. T. Baker silica gel (230ꢀ400 mesh). Melting points were determined
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by capillary method using MP70 Melting Point System (Mettler Toledo). H NMR (400 MHz)
and 13C NMR (100 MHz) were recorded on a Bruker Avance II 400 MHz spectrometer. The
chemical shifts (δ) are given in parts per million (ppm) relative to tetramethylsilane (TMS). IR
spectra were recorded on a Nicolet series II MagnaꢀIR 550 FTꢀIR spectrometer. ESIꢀMS spectra
were obtained using timeꢀofꢀflight detector on a MicroMass LCT mass spectrometer. HRMS
data were acquired on an Agilent Technologies 6530 AccurateꢀMass QꢀTOF LC/MS. HPLC was
performed with LabAlliance apparatus consisted of pumps (III Pump series), UVꢀVIS detector
(525 Dualwavelength) and injection valve (Rheodyne Model 7725i). Analytical HPLC was
carried out with a SUPELCOSIL LCꢀNH2ꢀNP HPLC column (5 ꢁm), 0.46x25 cm.
2-Methyl-1,4-naphthohydroquinone; Menadiol (4a). To a solution of menadione (S1, 10 g,
58 mmol) in EtOAc (70 mL) was added solution of Na2S2O4 (20 g, 115 mmol) in water (70 mL)
under argon atmosphere28. The reaction mixture was stirred vigorously at room temperature for
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20 min. The organic layer was separated, washed with water (2 x 40 mL), brine (1 x 40 mL) and
dried over anhydrous Na2SO4. After filtration, the solvent was removed under reduced pressure
to give 4a as a bright purple powder (9.6 g, yield 95%) which was used directly in the next step.
Mp 179ꢀ181 °C (181 °C35, 166 °C30). 1H NMR (400 MHz, DMSOꢀd6, ppm): δ 9.32 (s, 1H), 8.22
(s, 1H), 8.06 (d, J = 8.2 Hz, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.42ꢀ7.31 (m, 2H), 6.63 (s, 1H), 2.27
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(s, 3H). C NMR (100 MHz): δ 145.8, 141.7, 126.5, 124.9, 123.8, 123.5, 121.8, 121.7, 118.7,
111.1, 16.6. IR (KBr): ν 3270, 1603, 1205 cmꢀ1.
2-Methyl-1,4-dimethoxynaphthalene (4). To a solution of menadiol (4a, 9.54 g, 0.055 mol)
in anhydrous acetone (250 mL) were added K2CO3 (76 g, 0.55 mol) and Me2SO4 (27 mL, 0.28
mol) and the reaction mixture29 was stirred under reflux for 6 h. After cooling to room
temperature, the inorganic material was filtered off and the filtrate was concentrated under
reduced pressure. To the residue dissolved in Et2O (300 ml) 20% aqueous KOH solution (150
mL) was added and the mixture was stirred vigorously for 2 h. The organic layer was separated,
washed with water (1 x 100 mL), brine (1 x 100 mL) and dried over anhydrous Na2SO4. After
filtration, the solvent was evaporated in vacuo and the crude product was purified using DFC
(hexane) to obtain 4 as a yellow oil (9.35 g, yield 80%, after 2 steps). Rf = 0.54 (hexane/EtOAc,
9:1). 1H NMR (400 MHz, CDCl3, ppm): δ 8.21 (d, J = 8.4 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H),
7.55ꢀ7.51 (m, 1H), 7.46ꢀ7.42 (m, 1H), 6.62 (s, 1H), 3.98 (s, 3H), 3.88 (s, 3H), 2.47 (s, 3H).13C
NMR (100 MHz): δ 151.5, 147.0, 128.6, 126.4, 125.6, 125.2, 124.5, 122.2, 121.4, 106.8, 61.2,
55.6, 16.3 ppm. IR (CHCl3): ν 3011, 2938, 1598, 1508, 1462, 1266, 1121 cmꢀ1.
(E)-4-Chloro-2-methyl-1-phenylsulfonyl-2-butene (5). A mixture of PhSO2Cl (13 mL, 0.1
mol), isoprene (S2, 10 mL, 0.1 mol), CuCl (0.201 g, 2 mmol) and Et3N·HCl (0.419 g, 3 mmol)
in dry acetonitrile (6 mL) was stirred under reflux. After 3 h the reaction mixture was cooled to
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room temperature and an additional portion of isoprene (6 mL, 60 mmol) was added. The
reaction mixture was refluxed for 6 h, further stirred for 14 h at room temperature, and finally
for 3 h again under reflux. After evaporation, the residue was dissolved in chloroform (20 mL)
and 1% aqueous HCl solution (100 mL) was added. The organic phase was separated and water
layer was extracted with CH2Cl2 (3 x 15 mL). The combined organic layers were dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was treated with 2ꢀpropanol
and the crystals were collected by filtration, washed with 2ꢀpropanol and dried under reduced
pressure to give 5 as a white crystalline powder (14.68 g, 60%, purity by HPLC 99%). Rf = 0.24
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(hexane/EtOAc, 7:2); Mp 72ꢀ73 °C. H NMR (400 MHz, CDCl3, ppm): δ 7.87 (d, J = 7.4 Hz,
2H), 7.68ꢀ7.65 (m, 1H), 7.58ꢀ7.54 (m, 2H), 5.31 (tm, 1H), 3.97 (d, J = 7.8 Hz, 2H), 3.77 (s, 2H),
1.88 (s, 3H).13C NMR (100 MHz): δ 138.1, 133.8, 130.5, 129.8, 129.1, 128.5, 65.5, 39.3, 16.9
ppm26. IR (CHCl3): ν 3028, 1661, 1587, 1448, 1319, 1309, 1136 cmꢀ1.
(2'E)-2-(3'-Methyl-4'-phenylsulfonylbut-2'-enyl)-1,4-dimethoxy-3-methylnaphthalene
(2). To a solution of 4 (9.0 g, 44 mmol) and 5 (13.0 g, 53 mmol) in anhydrous 1,2ꢀ
dichloroethane (80 mL) SnCl4 (6.2 mL, 53 mmol) was slowly added at room temperature.31 The
reaction mixture was stirred for 5 h at 80 °C and cooled to room temperature. Then, water (20
mL) and CH2Cl2 (40 mL) were added. The organic phase was separated, washed with 10%
aqueous NH4Cl solution, dried over anhydrous Na2SO4, the drying agent was filtered off and the
filtrate was concentrated under reduced pressure. The residue was purified using DFC
(hexane/EtOAc, 7:2) to obtain an oily product (9.128 g). Further crystallization from methanol
afforded compound 2 as a white crystalline powder (5.480 g, 30%, purity by HPLC 99.6%). Rf
= 0.21 (hexane/EtOAc, 7:2); Mp (from methanol) 155.1ꢀ157.2 °C, 156ꢀ157 °C36; 1H NMR (400
MHz, CDCl3, ppm): δ 8.08ꢀ8.00 (m, 2H), 7.76ꢀ7.74 (m, 2H), 7.50ꢀ7.47 (m, 2H), 7.39ꢀ7.35 (m,
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1H), 7.31ꢀ7.27 (m, 2H), 5.03 (t, J = 6.6 Hz, 1H), 3.86 (s, 3H), 3.80 (s, 3H), 3.74 (s, 3H), 3.49 (d,
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J = 6.6 Hz, 2H), 2.22 (s, 3H), 2.02 (s, 3H) ppm; C NMR (100 MHz): δ 150.1, 149.9, 138.2,
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134.3, 133.3, 128.77, 128.75, 128.2, 127.6, 127.1, 126.2, 125.7, 125.4, 123.9, 122.2, 122.1, 66.0,
62.1, 61.3, 26.7, 17.1, 12.4 ppm36; IR (CHCl3): ν 3028, 1592, 1448, 1353, 1308, 1133 cmꢀ1.
(2E,6E)-3,7,11-Trimethyldodeca-2,6,10-trien-1-yl acetate; (E,E)-farnesyl acetate (6). To a
solution of E,Eꢀfarnesol (S3, 15 g, 67 mmol) in anhydrous pyridine (50 mL) acetic anhydride (30
mL) was added at 0 °C under argon atmosphere. The reaction mixture was stirred at room
temperature overnight. Upon completion (TLC control) the reaction mixture was poured into ice
water and extracted with EtOAc (3 x 60 mL). The organic layers were collected, washed with
10% HCl solution, 10% NaHCO3 solution and brine, dried over Na2SO4 and, after removal of the
drying agent, evaporated in vacuo. The crude product was purified using DFC (hexane) to give 6
as a light yellow oil (16.94 g, 95%). Rf = 0.70 (hexane/EtOAc, 7:2).1H NMR (400 MHz, CDCl3,
ppm): δ 5.36–5.32 (m, 1H), 5.11–5.07 (m, 2H), 4.59 (d, J = 7.1 Hz, 2H), 2.13ꢀ1.96 (m, 8H), 2.05
(s, 3H) 1.71 and 1.68 (2s, 6H), 1.60 (s, 6H). 13C NMR (100 MHz): δ 171.0, 142.2, 135.4, 131.2,
124.3, 123.6, 118.3, 61.3, 39.6, 39.5, 26.7, 26.1, 25.6, 21.0, 17.6, 16.4, 15.9.33 IR (CHCl3): ν
2968, 1737, 1228 cmꢀ1.
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(2E,6E,10E)-12-Hydroxy-3,7,11-trimethyldodeca-2,6,10-trien-1-yl acetate; (E,E,E)-12-
hydroxyfarnesyl acetate (7). Tertꢀbutyl hydroperoxide (31 mL, 70 wt.% in H2O) was added to
a stirred suspension of SeO2 (710 mg, 6.4 mmol) and salicylic acid (884 mg, 6.4 mmol) in
CH2Cl2 (120 mL). The stirring was continued for 30 min at room temperature, then cooled to 0
°C and the solution of 6 (16.94 g, 76 mmol) in CH2Cl2 (12 mL) was added dropwise. The
reaction was carried out for 24 h at room temperature and concentrated under vacuo. The residue
dissolved in Et2O (120 mL) was washed with 5% aqueous KOH solution and brine, dried over
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anhydrous Na2SO4. The drying agent was filtered off and the clear solution was evaporated
under reduced pressure. The oily residue was dissolved in a mixture of MeOH (6 mL) and THF
(114 mL) and NaBH4 (4.9 g, 130 mmol) was added portionwise at ꢀ10°C. After stirring for 30
min the reaction mixture was poured into saturated aqueous NH4Cl solution (120 mL) and
extracted with EtOAc (3x100 mL). Combined organic extracts were washed with brine, dried
over anhydrous Na2SO4, and, after removal the drying agent, evaporated to dryness. The residue
was purified by FC (hexane/EtOAc, 88:12), to yield 7 as a colorless oil (5.2 g, yield 29%). Rf =
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0.27 (hexane/EtOAc, 7:2); H NMR (400 MHz, CDCl3, ppm): δ 5.41ꢀ5.33 (m, 2H), 5.12ꢀ5.09
(m, 1H), 4.59 (d, J = 7.1 Hz, 2H), 3.99 (s, 2H), 2.16ꢀ2.00 (m, 8H), 2.05 (s, 3H), 1.71 (s, 3H),
1.67 (s, 3H), 1.61 (s, 3H). 13C NMR (100 MHz): δ 171.1, 142.2, 135.1, 134.7, 125.9, 123.9,
118.3, 68.9, 61.4, 39.4, 39.2, 26.13, 26.10, 21.0, 16.4, 16.0, 13.6 ppm; IR (CHCl3): ν 3604,
3467, 2927, 1728, 1610, 1239 cmꢀ1.
(2E,6E,10E)-3,7,11-Trimethyl-12-(phenylsulfonyl)dodeca-2,6,10-trien-1-yl acetate (8). To
a stirred solution of 7 (5.2 g, 18.5 mmol) in dry THF (5 mL) PBr3 (0.8 mL, 8.5 mmol) was added
at 0 °C under argon atmosphere. After 3 h the reaction mixture was quenched by pouring to ice
water and extracted with Et2O (3 x 50 mL). Combined organic extracts were washed with 5%
aqueous NaHCO3 solution and brine, dried over anhydrous Na2SO4, filtered through a filter
paper and evaporated under vacuum to yield crude bromide 7a as a colorless oil (5.08 g, yield
80%) which was used directly in the next step without further purification.
To a solution of 7a (5.08 g, 14.8 mmol) in anhydrous DMF (50 mL) sodium benzenesulfinate
(PhSO2Na) (3.15 g, 19.2 mmol) was added and the resulting suspension was stirred at room
temperature in the dark. After 18 h the reaction mixture was poured into water (100 mL) and
extracted with Et2O (3 x 100 mL). The combined organic extracts were washed with saturated
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aqueous NH4Cl solution and brine, dried over anhydrous Na2SO4 and filtered. Solvent was
removed under vacuum at 40 °C and the crude product was purified by DFC (hexane/EtOAc,
7:2) to give 8 as a colorless oil (5.25 g, yield 70% after two steps). Rf = 0.41 (hexane/EtOAc =
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7:2). H NMR (400 MHz, CDCl3, ppm): δ 7.86ꢀ7.84 (m, 2H), 7.66ꢀ7.62 (m, 1H), 7.56ꢀ7.52 (m,
2H), 5.35ꢀ5.31 (m, 1H), 5.07ꢀ4.99 (m, 2H), 4.58 (d, J = 7.1 Hz, 2H), 3.72 (s, 2H), 2.05 (s, 3H),
2.09ꢀ2.01 (m, 8H), 1.76 (s, 3H), 1.70 (s, 3H), 1.54 (s, 3H) ppm; 13C NMR (100 MHz): δ 171.1,
142.0, 138.5, 136.0, 134.6, 133.4, 128.8, 128.5, 124.1, 123.2, 118.3, 66.2, 61.3, 39.4, 38.5, 26.9,
26.1, 21.0, 16.7, 16.4, 15.9 ppm; IR (CHCl3): ν 2930, 1728, 1667, 1145 cmꢀ1.
(2E,6E,10E,14E,18E)-3,7,11,15,19,23-Hexamethyl-12-(phenylsulfonyl)tetracosa-
2,6,10,14,18,22-hexaen-1-ol (9). To a stirred solution of 8 (5.21 g, 12.9 mmol) in dry THF (40
mL) and DMF (10 mL) a suspension of tꢀBuOK (1.594 g, 14.2 mmol) in anhydrous THF was
added at ꢀ78 °C. The stirring was continued at ꢀ78°C for 2.5 h and a solution of trans,transꢀ
farnesyl bromide (4.051 g, 14.2 mmol, purity: 95%) in THF (10 mL) was added dropwise. The
reaction was carried out at ꢀ78 °C for 2 h and at room temperature overnight. Then the reaction
mixture was poured to saturated aqueous NH4Cl solution (100 mL) and extracted with Et2O (3 x
50 mL). Combined organic phases were washed with brine, dried over anhydrous Na2SO4,
filtered through a filter paper and evaporated under reduced pressure. To the residue dissolved in
MeOH (20 mL) was added 1M NaOH solution to pH 12 and stirred at room temperature for 1 h.
After removal of MeOH under vacuum water was added and the reaction mixture was extracted
with Et2O (3 x 100 mL). Combined organic extracts were washed with brine, dried over
anhydrous Na2SO4, filtered and evaporated to dryness. The crude product was purified by DFC
(hexane/EtOAc, 7:2) to afford 9 as a light yellow oil (3.61 g, yield 50%). Rf = 0.23
1
(hexane/EtOAc, 7:2). H NMR (400 MHz, CDCl3, ppm): δ 7.82ꢀ7.51 (3m, 5H), 5.42ꢀ5.38 (m,
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1H), 5.08ꢀ4.99 (m, 4H), 4.90ꢀ4.86 (m, 1H), 4.15 (d, J = 6.9 Hz, 2H), 3.47 (dd, J = 11.6, 3.9 Hz,
1H), 2.82ꢀ2.78 (m, 1H), 2.62ꢀ2.60 (m, 1H), 2.08ꢀ1.92 (m, 16H), 1.67 (s, 6H), 1.64 (2s, 6H), 1.58
(s, 3H), 1.56 (s, 3H), 1.52 (s, 3H) ppm; 13C NMR (100 MHz): δ 139.6, 138.4, 138.2, 135.6,
135.2, 134.7, 133.2, 131.3, 128.79, 128.75, 126.6, 124.3, 124.1, 123.8, 123.4, 118.8, 74.1, 59.4,
39.7, 39.6, 39.4, 38.6, 26.8, 26.5, 26.3, 25.7, 24.1, 17.7, 16.3, 16.0, 15.9, 13.8 ppm; IR (CHCl3):
ν 3607, 3532, 2926, 1667, 1586, 1144 cmꢀ1. ESI-MS (m/z): 589.1 [MNa]+. HRMS (ESI) calcd
for C36H55O3S [MH]+ 567.3866, found 567.3877 (1.9 ppm error).
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((2E,6E)-3,7,11-Trimethyldodeca-2,6,10-trien-1-yl)benzenesulfonate (8a). Compound 8a
was prepared analogously to the procedure described above for 8 using trans,transꢀfarnesol (1 g,
4.5 mmol), PBr3 (0.2 mL, 2.1 mmol), dry THF (5 mL) followed by using PhSO2Na (0.755 g, 4.6
mmol) and dry DMF (10 mL). The crude product was purified by FC (hexane/EtOAc, 95:5) to
give 8a as a colorless oil (1.23 g, 79%). Rf = 0.53 (hexane/EtOAc = 7:2).1H NMR (400 MHz,
CDCl3, ppm): δ 7.89ꢀ7.86 (m, 2H), 7.66ꢀ7.62 (m, 1H), 7.56ꢀ7.52 (m, 2H), 5.20 (t, J = 8.0 Hz,
1H), 5.10ꢀ4.04 (m, 2H), 3.81 (d, J = 8.0 Hz, 2H), 2.07ꢀ2.1.96 (m, 8H), 1.68 (s, 6H), 1.60 (s, 3H),
1.59 (s, 3H), 1.32 (s, 3H) ppm; 13C NMR (100 MHz): δ 146.4, 138.7, 135.7, 133.5, 131.4, 128.9,
128.5, 124.20, 123.3, 110.3, 56.1, 39.67, 39.66, 26.7, 26.2, 25.7, 17.7, 16.2, 16.0 ppm; IR
(CHCl3): ν 2928, 1448, 1307, 1150, 1085 cmꢀ1.
(2E,6E,10E,14E,18E)-3,7,11,15,19,23-hexamethyl-13-(phenylsulfonyl)tetracosa-
2,6,10,14,18,22-hexaen-1-ol (9a). Bromide 7a was freshly prepared from alcohol 7 (1 g, 3.57
mmol) as described previously. To a stirred solution of 8a (1.09 g, 3.14 mmol) in dry THF (12
mL) and HMPA (3 mL) nꢀBuLi (2.0 mL, 3.2 mmol, 1.6 M) was added at ꢀ78 °C. The stirring
was continued at ꢀ78°C for 1.5 h and a solution of 7a (0.98 g, 2.85 mmol) in dry THF was
added. After 5 h the reaction mixture was allowed to warm up to 0 °C, poured to saturated
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NH4Cl solution (10 mL) and extracted with Et2O (3 x 10 mL). Combined organic extracts were
washed with brine, dried over anhydrous Na2SO4, the drying agent was filtered off and solvent
was evaporated under reduced pressure. To the residue dissolved in MeOH (10 mL) catalytic
amount of MeONa was added and the reaction mixture was stirred at room temperature for 2 h.
The solvent was evaporated under vacuum and the residue was purified by FC (hexane/EtOAc,
75:25) to give 9a as a light yellow oil (0.84 g, 52%). 1H NMR (400 MHz, CDCl3, ppm): δ 7.86ꢀ
7.84 (m, 2H), 7.64ꢀ7.60 (m, 1H), 7.59ꢀ7.50 (m, 2H), 5.42 (t, J = 6.9 Hz, 1H), 5.15 (t, J = 6.8 Hz,
1H), 5.10ꢀ5.05 (m, 3H), 4.93 (d, J = 10.4 Hz, 2H), 4.16 (d, J = 6.8 Hz, 2H), 3.89 (dt, J = 10.9
and 3.0 Hz, 1H), 2.89 (d, J = 12.6 Hz, 1H), 2.29 (dd, J = 13.3 and 11.5 Hz, 1H), 2.05ꢀ1.94 (m,
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16H), 1.69 (s, 6H), 1.61 (s, 3H), 1.59 (s, 3H), 1.57 (s, 3H), 1.53 (s, 3H), 1.18 (s, 3H) ppm; C
NMR (100 MHz): δ 145.0, 139.6, 138.0, 135.6, 135.0, 133.3, 131.4, 129.8, 129.2, 128.7, 128.2,
124.2, 124.0, 123.5, 123.4, 117.3, 63.6, 59.4, 39.72, 39.70, 39.5, 39.3, 37.3, 26.7, 26.6, 26.4,
26.2, 25.7, 17.7, 16.33, 16.27, 15.9 ppm; IR (CHCl3): ν 3606, 3515, 2927, 1666, 1447, 1145,
1084 cmꢀ1. ESI-MS (m/z): 589.1 [MNa]+; HRMS (ESI) calcd for C36H55O3S [MH]+ 567.3866,
found 567.3875 (1.6 ppm error).
(2E,6E,10E,14E,18E)-3,7,11,15,19,23-Hexamethyltetracosa-2,6,10,14,18,22-hexaen-1-ol;
all-trans-hexaprenol (10). To a solution of 9 (3.5 g, 6.2 mmol) in dry THF (20 mL) Pd(dppe)Cl2
(345 mg, 0.6 mmol) and LiEt3BH (19 mL, 19 mmol, 1M solution in THF) were added at 0 °C
under argon atmosphere. The reaction mixture was stirred at room temperature until completion
as monitored by TLC (usually up to 2 h). The mixture was poured into 20% NH4Cl (40 mL) and
thoroughly extracted with EtOAc (3 x 30 mL). Combined organic layers were washed with
brine, dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. The crude
product was purified by DFC (hexane/EtOAc, 70:5 to 70:20) to give 10 as a colorless oil (2.0 g,
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yield 76%, HPLC purity: 91%). Rf = 0.47 (hexane/EtOAc, 7:2); H NMR (400 MHz, CDCl3,
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ppm): δ 5.44ꢀ5.41 (m, 1H), 5.14ꢀ5.09 (m, 5H), 4.15 (d, J = 7.0 Hz, 2H), 2.10ꢀ1.98 (m, 20H),
1.69 and 1.61 (2s, 21H); 13C NMR (100 MHz): δ 139.7, 135.3, 134.94, 134.86, 134.8, 131.2,
124.4, 124.23, 124.15, 123.7, 123.4, 59.3, 39.7, 39.5, 26.7, 26.65, 26.63, 26.3, 25.6, 17.6, 16.2,
15.97, 15.95; IR (CHCl3): ν 3611, 3452, 2918, 1667, 1449, 1383 cmꢀ1; ESI-MS (m/z): 449
[MNa]+; HRMS (ESI) calcd for C30H51O [MH]+ 427.3934, found 427.3943 (2.1 ppm error).
Following the previous procedure for 9, the reaction of 9a (0.83 g, 1.5 mmol) and LiEt3BH
(5 mL, 5.0 mmol, 1M solution in THF) under Pd(dppe)Cl2 (92 mg, 0.16 mmol) catalysis in THF
(7 mL) at 0 °C afforded 10 as a colorless oil (0.456 g, 73%). The NMR spectra were identical
with those disclosed above.
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Hexaprenyl bromide (3). To a stirred solution of 10 (2.0 g, 4.69 mmol) in anhydrous THF
(25 mL) PBr3 (0.2 mL, 2.13 mmol) was added at 0 °C under argon atmosphere. After 3 h the
reaction mixture was poured into ice water (50 mL) and extracted with Et2O (3 x 50 mL).
Combined organic extracts were washed with 5% NaHCO3, brine and water, dried over
anhydrous Na2SO4, and, after removal of inorganic material, evaporated under vacuum. The
crude bromide 3 (2.27 g, 87%, colorless oil) was used in the next step without further
purification.
2-((2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-Heptamethyl-4-(phenylsulfonyl)octacosa-
2,6,10,14,18,22,26-heptaen-1-yl)-1,4-dimethoxy-3-methylnaphthalene (11). t-BuOK (493 mg,
4.4 mmol) was added to a stirred solution of 2 (1.52 g, 3.7 mmol) and 3 (2.27 g, 4.1 mmol) in
THF (18 mL) and DMF (2 mL) at ꢀ20 ºC. The stirring was continued at ꢀ20 ºC until completion
of the reaction (TLC control). After 1 h the reaction mixture was allowed to warm up to room
temperature and poured to saturated NH4Cl solution (40 mL). Crude product was extracted with
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EtOAc (3 x 20 mL). The combined organic extracts were washed with water, dried over
anhydrous Na2SO4 and, after removal of the drying agent, evaporated to dryness under reduced
pressure. The residue was purified by FC (hexane/EtOAc, 7:2) to obtain compound 11 as a
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bright yellow oil (2.475 g, 82%). Rf = 0.32 (hexane/EtOAc, 7:2); H NMR (400 MHz, CDCl3,
ppm): δ 8.06ꢀ7.99 (m, 2H), 7.75 (d, J = 7.0 Hz, 2H), 7.49ꢀ7.31 (3m, 5H), 5.14ꢀ5.03 (m, 6H),
4.89 (t, J = 6.9 Hz, 1H), 3.84 (s, 3H), 3.74 (s, 3H), 3.52ꢀ3.38 (m, 3H), 2.85ꢀ2.62 (2m, 2H), 2.16
(s, 3H), 2.07ꢀ1.96 (m, 20H), 1.91 (s, 3H), 1.69 (s, 3H), 1.61 (s, 12H), 1.60 (s, 3H), 1.57 (s, 3H)
ppm; 13C NMR (100 MHz): δ 150.1, 149.8, 138.7, 137.9, 135.3, 135.0, 134.9, 134.3, 133.1,
131.2, 129.0, 128.6, 127.6, 127.4, 127.2, 126.3, 125.6, 125.4, 124.4, 124.2, 124.1, 123.7, 122.2,
122.1, 118.5, 73.9, 62.0, 61.3, 39.7, 30.3, 29.7, 26.8, 26.7, 26.73, 26.71, 26.67, 26.64, 26.54,
26.49, 25.7, 23.9, 17.7, 16.3, 16.1, 16.02, 15.99, 13.9, 12.4; IR (CHCl3): ν 2930, 2855, 1592,
1353, 1145 cmꢀ1; ESI-MS (m/z): 841.3 [MNa]+; HRMS (ESI) calcd for C54H75O4S [MH]+
819.5381, found 819.5392 (1.3 ppm error).
Preparation of vitamin MK-7 (1).
Desulfonation. Following the previous procedure for 10, the reaction of 11 (1.7 g, 2.1 mmol),
LiEt3BH (4.6 mL, 4.6 mmol, 1M in THF) and Pd(dppe)Cl2 (121 mg, 0.21 mmol) catalyst in dry
THF (5 mL) at 0 ºC for 4 h afforded 12 as a colorless oil (1.094 g, 78%), which was used
immediately in the next step without further purification.
Oxidation. A solution of ammonium cerium(IV) nitrate (CAN) (2.19 g, 4 mmol) in CH3CN
(12 mL) and H2O (2 mL) was slowly added to a stirred solution of 12 (1.094 g, 1.6 mmol) in a
mixture of CH3CN (6 mL) and CH2Cl2 (6 mL) at 0°C. After 45 min (TLC control) the reaction
mixture was poured into ice cold water (100 mL) and extracted with CH2Cl2 (3 x 50 mL). The
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combined organic layers were washed with brine and water, dried over anhydrous Na2SO4,
filtrated and evaporated under reduced pressure at 40 °C. Crude product was purified by DFC
(hexane/CH2Cl2, 5:1) to obtain 1 as a yellow oil (0.753 g, yield 72%, HPLC purity: 99.4%).
Further crystallization from ethyl acetate and ethanol afforded vitamin MKꢀ7 (1) as a yellow
powder (0.64 g, purity by HPLC: 99.9%). Rf = 0.40 (hexane/EtOAc 7:2); Mp 51.5ꢀ53.5 ˚C
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(54.68 °C34); H NMR (CDCl3): δ 8.12ꢀ8.07 (m, 2H), 7.72ꢀ7.67 (m, 2H), 5.14ꢀ5.01 (m, 7H),
3.38 (d, J = 6.9 Hz, 2H), 2.20 (s, 3H), 2.08ꢀ1.96 (m, 24H), 1.80 (s, 3H), 1.69 (s, 3H), 1.60 (s,
13
12H), 1.57 (2s, 6H) ppm; C NMR (CDCl3): δ 185.45, 184.51, 146.18, 143.35, 137.57, 135.23,
134.94, 134.91, 134.88, 133.31, 133.25, 132.22, 132.18, 131.23, 126.31, 126.19, 124.42, 124.28,
124.16, 123.85, 119.09, 39.72, 39.69, 26.78, 26.71, 26.68, 26.52, 26.01, 25.68, 17.67, 16.43,
16.01, 16.00, 12.66; IR (KBr): ν 2963, 1660, 1618, 1594, 1295 cmꢀ1; ESI-MS (m/z): 671.9
[MNa]+; HRMS (ESI) calcd for C46H65O2 [MH]+ 649.4979, found 236.1283 (1.5 ppm error).
ASSOCIATED CONTENT
1
13
Supporting Information. H and C NMR spectra for all intermediates and the final product
(vitamin MKꢀ7). IR, MS for compound 1 and HPLC chromatograms for compounds 2, 3 and 1.
AUTHOR INFORMATION
Corresponding Authors
*Phone: +48 85 7388086. Eꢀmail: wit@uwb.edu.pl
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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The authors would like to thank Mrs J. Maj for HPLC analysis and Dr L. Siergiejczyk for
recording NMR spectra. The research was supported in part by Europharma Alliance, Wrocław,
Poland.
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