Checked by Gilles Chambournier and David J. Hart.
1. Procedure
A.
3,5-Dinitrobenzoate
of vitamin D2
(
3). A
2-L photo-reaction vessel equipped with a quartz immersion well, a
thermometer, an
argon-inlet tube, a
mineral oil outlet-bubbler, a
mechanical stirrer, and supported in an adequately sized
Dewar
(Note 1), is charged with
13.5 g (34 mmol) of ergosterol
(
1)
(Note 2),
1.31 g (6.8 mmol) of ethyl p-dimethylaminobenzoate
, and
1.7 L of tert-butyl methyl ether (tert-BuOMe)
(Note 3). The mixture is stirred at room temperature overnight with gentle bubbling of
argon
(Note 4). A
450-watt Hanovia medium-pressure mercury lamp is inserted into the well, through which a fast stream of water is continuously passed
(Note 5). The solution is cooled in a dry
ice-ethanol bath and stirred vigorously
(Note 6). When the temperature of the solution reaches 0°C, the lamp is turned on, and irradiation is continued at 0°C to −20°C for 4 hr
(Note 7). The lamp is turned off, a solution of
75 mg (0.34 mmol) of 9-acetylanthracene
(Note 8) in
3 mL of tert-BuOMe is added to the solution, and a
uranium filter
(Note 9) is inserted into the arc housing
(Note 10). After 10 min, the lamp is started again, and the mixture is irradiated at 0°C to −20°C for 1 hr through the uranium filter
(Note 11). The cold, pale yellow solution of
pre-vitamin 2
thus obtained
(Note 12) is transferred to a
3-L, round-bottomed flask and concentrated at 20-25°C under reduced pressure. The residue is transferred again to a
500-mL, round-bottomed flask using tert-BuOMe to allow quantitative transfer. The solution is concentrated and dried at room temperature under high vacuum (1 mm) for 30 min to give approximately
15.4 g of a yellow resin. The flask is filled with
argon and equipped with a
magnetic stirrer.
Methanol (100 mL) is added, and the mixture is shaken to give a stirrable suspension. The suspension is then stirred for 45 min at room temperature and stored in a freezer overnight. After the mixture is stirred at −30°C
(Note 13) for 30 min, it is quickly filtered through a
60-mL sintered-glass funnel of coarse porosity. The collected solid is washed with
20 mL of cold methanol
(Note 14).
Ergosterol (1.83 g, 14%
recovery, mp
145-151°C, 99.4% pure) is recovered by washing this solid with absolute
ethanol (30 mL at room temperature). The filtrate and cold-methanol wash are transferred to a 500-mL, round-bottomed flask equipped with a magnetic stirrer, an argon-inlet tube, and a
reflux condenser. The flask is flushed with
argon, and the orange solution is heated under reflux for 6 hr
(Note 15) and then stirred at 35-40°C overnight
(Note 16). The mixture is concentrated at 30°C under reduced pressure, and the residual
methanol is removed by coevaporation with
50 mL of toluene
at 30°C to give approximately
14.6 g of an orange-tan oil. The flask is filled with
argon and then equipped with a magnetic stirrer. The residue is dissolved in
40 mL of pyridine
, and the solution is cooled in an
ice-water bath. Solid
3,5-dinitrobenzoyl chloride (9.0 g, 39 mmol)
(Note 17) is added in small portions over 5 min followed by
20 mL of pyridine
to rinse the walls of the flask, and the mixture is stirred at 0°C for 20 min. The very thick suspension obtained is shaken and then allowed to stand at 0°C for a further 20 min, whereupon
methanol (30 mL) is added to the cold mixture. The mixture is allowed to stand at 0°C for 5 min, and then it is shaken for about 5 min to give a stirrable suspension. After the orange suspension is stirred at 0°C for 1.5 hr, it is diluted by the dropwise addition of
150 mL of methanol
over 15 min and stirred at 0°C for another hour. The yellow solid is collected by filtration, washed with
50 mL of ice-cold methanol
, and dried at room temperature under high vacuum for 2 hr. The yellow-orange solid is transferred to a
250-mL, round-bottomed flask equipped with a magnetic stirrer and an argon-inlet tube. The flask is flushed with
argon, and the solid is suspended in
50 mL of absolute ethanol
. The suspension is stirred at room temperature for 15 min and at 0°C for 45 min. The solid is collected by filtration, washed with
20 mL of cold methanol
, and dried at room temperature under high vacuum overnight to give
8.3-10.1 g (
41-50%) of
3 as a yellow solid, mp
139-141°C (lit.
2
3
147-149°C)
(Note 18) and
(Note 19).
2. Notes
1.
The reactor is available from Ace Glass Inc. [
reaction vessel (#7851-17),
immersion well (#7854-28, 290 mm),
Teflon bearing (#8066-24), and
stirring shaft (#8068-303)]. It is similar to that shown in
Figure 1 (Org. Synth., Coll. Vol. V
1973, p.529) with an additional stirring chamber. The submitter used a
4-mm I.D. tube for an argon-inlet in order to avoid clogging; the tube should reach near to the bottom of the vessel. The checkers used a
20 × 45-cm (ID × height) Dewar, available from Cole-Parmer Instrument Co. #H-03774-54).
2.
Ergosterol (1), obtained from Aldrich Chemical Company, Inc.
(mp
134-142°C, ε 8,030 at 282 nm in EtOH), was purified before use as follows:
24.4 g of 1
was suspended in
200 mL of ethanol (EtOH), and the mixture was stirred at room temperature for 3 hr prior to filtration. The collected solid was washed with
40 mL of EtOH and dried under high vacuum (1.0 mm) to give
19.3 g (79% recovery) of 1
as a white solid (mp
147-153°C, ε 11,900 at 282 nm in EtOH). The submitter observed that when
1 purchased from Kaneka Co. (mp
147-153°C, ε 11,560 at 282 nm in EtOH) was used as received, a better quality of vitamin D
2 (
4) (mp
114-115°C, 99.8% pure) was obtained in a better overall yield of
48% (
55% based on the recovered
ergosterol). The checkers used
ergosterol obtained from Acros Organics
(mp
156-158°C), which was purified as described above (mp
154-158°C).
3.
Ethyl p-dimethylaminobenzoate and tert-butyl methyl ether (HPLC grade) were obtained from Aldrich Chemical Company, Inc.
, and used as received.
4.
The overnight stirring with bubbling of
argon to remove
oxygen is probably too long, but was done simply for convenience, thereby allowing the irradiation to be carried out the next day. The checkers found that 4 hr was sufficient.
5.
Water flow must be very fast to avoid freezing, which could result in breakage of the photochemical reactor and generation of a hazardous situation. Water flow should be monitored continuously during the course of the reaction.
6.
Efficient stirring is necessary to achieve relatively homogeneous temperature distribution throughout the reaction mixture. The submitter recommends that the mixture be kept below 0°C to prevent thermal isomerization of
1 to vitamin D
2, which produces a variety of photoproducts upon irradiation.
7.
The checkers monitored the reaction temperature at 5-min intervals and added dry ice to the bath each time the temperature approached 0°C. Approximately 30 pounds of dry ice are required over the 4-hr irradiation period. Using
1H NMR analyses, the submitter judged the conversion to be 80-85% after 4 hr of irradiation with the apparatus described. After 3 hr, a 1:2:1 mixture of
1:
2:tachy-isomer (see Discussion) was obtained. The diagnostic peaks in the
1H NMR spectra are listed in Table. The R
f values on silica gel TLC using 1:4 EtOAc-hexane are as follows:
1 (0.29),
4 (0.34), tachy-isomer (0.34),
2 (0.42),
ethyl p-dimethylaminobenzoate (0.47), and
9-acetylanthracene (0.53), using short wave UV detection.
TABLE
THE CHEMICAL SHIFTS OF DIAGNOSTIC PEAKS IN 1H NMRa
|
|
|
18-CH
3
|
3-H
|
olefinic protons
|
|
|
Ergosterol (1)
|
0.62 (s)
|
3.62 (m)
|
5.39 (m), 5.57 (m)
|
|
Pre-isomer (2)
|
0.71 (s)
|
3.92 (m)
|
5.48 (m), 5.67 (d, 11 Hz), 5.93 (d, 11 Hz)
|
|
Tachy-isomer
|
0.69 (s)
|
3.92 (m)
|
5.67 (m), 6.00 (d, 16 Hz), 6.71 (d, 16 Hz)
|
|
a The chemical shifts are reported in ppm relative to CHCl3 (7.25) in CDCl3 as an internal standard.
|
8.
9-Acetylanthracene was purchased from Aldrich Chemical Company, Inc.
and used as received.
9.
A
cylindrical uranium filter (31-mm O.D., 2.5-mm thickness) was obtained from Houde Glass Co., Inc.
10.
Caution! The lamp is very hot. The lamp should be allowed to cool for 10 min before restarting to prevent damage.
11.
Based on
1H NMR analyses, the photosensitized isomerization of the tachy-isomer into
2 was complete after 40 min of irradiation (see Discussion).
12.
The submitter recommends that pre-isomer (
2) and vitamin D
2 (
4) not be exposed to air at room temperature for more than 30 min, since these compounds are relatively easily oxidized by air.
13.
The checkers used a dry
ice-ethylene glycol bath at −25°C.
14.
The checkers cooled the
methanol to −70°C in a dry
ice-acetone bath.
15.
After 3 hr of reflux, the ratio of
2 to
4 was ca. 1:3, whereas after 6 hr, it reached about 1:5.
16.
At this point, the ratio of
2 to
4 was greater than 1:6.
17.
3,5-Dinitrobenzoyl chloride was purchased from Aldrich Chemical Company, Inc.
and
pyridine (A.C.S. certified) was obtained from Fisher Scientific Co.
They are used as received.
18.
The elemental analysis and spectral properties of
3 are as follows: Anal. Calcd for C
35H
46N
2O
6: C, 71.16; H, 7.85; N, 4.74. Found: C, 71.05; H, 7.89; N, 4.58; IR (KBr) cm
−: 1733, 1546, 1342
;
1H NMR (CDCl
3) δ: 0.56 (s, 3 H), 0.82 (d, 3 H, J = 6.5), 0.84 (d, 3 H, J = 6.5), 0.92 (d, 3 H, J = 6.8), 1.02 (d, 3 H, J = 6.6), 1.25-2.17 (m, 16 H), 2.32 (m, 1 H), 2.50 (m, 1 H), 2.59 (dd, 1 H, J = 12.2 and 6.8), 2.73 (dd, 1 H, J = 12.2 and 4.5), 2.80 (dd, 1 H, J = 8.8 and 3.5), 4.91 (bs, 1 H), 5.13 (bs, 1 H), 5.20 (m, 2 H), 5.31 (m, 1 H), 6.06 (d, 1 H, J = 11.1), 6.28 (d, 1 H, J = 11.1), 9.13 (d, 2 H, J = 2.1), 9.22 (t, 1 H, J = 2.1)
;
13C NMR (CDCl
3) δ: 12.2 (q), 17.5 (q), 19.5 (q), 19.8 (q), 21.0 (q), 22.1 (t), 23.5 (t), 27.7 (t), 28.9 (t), 31.7 (t), 32.0 (t), 33.0 (d), 40.3 (d and t), 41.9 (t), 42.7 (d), 45.8 (s), 56.3 (d), 74.8 (d), 113.2 (t), 117.1 (d), 122.1 (d), 123.0 (d), 129.3 (d), 131.9 (d), 133.1 (s), 134.3 (s), 135.4 (d), 143.1 (s), 143.8 (s), 148.5 (s), 161.8 (s)
(one doublet was not observed).
19.
The submitter indicates that HPLC analysis of the product shows its purity to be 97.3% (with 0.3% of
ergosteryl 3,5-dinitrobenzoate). HPLC conditions for this unchecked analysis are as follows: column: Chromegasphere SI-60 (3μ,15-cm × 5-mm) (purchased from ES Industries); mobile phase: 5% EtOAc in
heptane (1 mL/min); detection: 275 nm. The retention times of
3 and
ergosteryl 3,5-dinitrobenzoate are 4.90 and 3.83 min, respectively. Those of the other impurities are 4.20, 4.55, 6.17, and 8.67 min. The checkers observed traces of
pyridine in the
1H NMR spectrum of the product.
20.
The checkers cooled this solution in an ice-water bath.
21.
The elemental analysis and the spectral properties of
4 are as follows: Anal. Calcd for C
28H
44O: C, 84.79; H, 11.18. Found: C, 84.89; H, 11.17. UV (EtOH) λ
max 264 nm (ε 18,450);
1H NMR (CDCl
3) δ: 0.55 (s, 3 H), 0.82 (d, 3 H, J = 6.5), 0.84 (d, 3 H, J = 6.5), 0.91 (d, 3 H, J = 6.5), 1.01 (d, 3 H, J = 6.7), 1.2-2.5 (m, 19 H), 2.57 (dd, 1 H, J = 11.6 and 3.5), 2.81 (broad d, 1 H, J = 9.0), 3.95 (m, 1 H), 4.82 (bs, 1 H), 5.04 (bs, 1 H), 5.20 (m, 2 H), 6.05 (d, 1 H, J = 11.2), 6.23 (d, 1 H, J = 11.2)
; The OH
hydrogen was not observed;
13C NMR (CDCl
3) δ: 12.2 (q), 17.5 (q), 19.5 (q), 19.8 (q), 21.0 (q), 22.1 (t), 23.4 (t), 27.7 (t), 28.9 (t), 31.8 (t), 33.0 (d), 35.0 (t), 40.3 (d and t), 42.7 (d), 45.6 (s), 45.8 (t), 56.3 (d), 69.1 (d), 112.3 (t), 117.4 (d), 122.3 (d), 131.8 (d), 134.9 (s), 135.5 (d), 142.1 (s), 144.0 (s)
(one doublet was not observed).
22.
The submitter indicates that HPLC analysis of the product shows the purity to be 99.7% (with 0.1% of
ergosterol). The HPLC conditions for this unchecked analysis are as follows:
column: Chromegasphere SI-60 (3μ, 15-cm × 5-mm) (purchased from ES Industries); mobile phase: 5% EtOAc in
heptane (2 mL/min); detection: 275 nm. The retention times of
1 and
4 are 19.54 and 16.27 min, respectively. The checkers observed signals due to a trace olefinic contaminant in the
1H NMR of the product at δ 6.47 and 5.95.
The procedures in this article are intended for use only by persons with prior training in experimental organic chemistry. All hazardous materials should be handled using the standard procedures for work with chemicals described in references such as "Prudent Practices in the Laboratory" (The National Academies Press, Washington, D.C., 2011 www.nap.edu). All chemical waste should be disposed of in accordance with local regulations. For general guidelines for the management of chemical waste, see Chapter 8 of Prudent Practices.
These procedures must be conducted at one's own risk. Organic Syntheses, Inc., its Editors, and its Board of Directors do not warrant or guarantee the safety of individuals using these procedures and hereby disclaim any liability for any injuries or damages claimed to have resulted from or related in any way to the procedures herein.
3. Discussion
Appendix
Chemical Abstracts Nomenclature (Collective Index Number);
(Registry Number)
Vitamin D2: Ergocalciferol (8);
9,10-Secoergosta-5,7,10 (19),
22-tetraen-3-ol, (3β)- (9); (50-14-6)
Ergosterol (8);
Ergosta-5,7,22-trien-3-ol, (3β)- (9); (57-87-4)
Vitamin D2
3,5-dinitrobenzoate:
Ergocalciferol, 3,5-dinitrobenzoate (8,9); (4712-11-2)
Ethyl p-dimethylaminobenzoate:
Benzoic acid, p-(dimethylamino)-, ethyl ester (8);
Benzoic acid, 4-(dimethylamino)-, ethyl ester (9); (10287-53-3)
tert-Butyl methyl ether:
Ether, tert-butyl methyl (8);
Propane, 2-methoxy-2-methyl- (9); (1634-04-4)
9-Acetylanthracene:
Ketone, 9-anthryl methyl (8);
Ethanone, 1-(9-anthracenyl)- (9); (784-04-3)
3,5-Dinitrobenzoyl chloride:
Benzoyl chloride, 3,5-dinitro- (8,9); (99-33-2)
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