hexane, and stirred until the temperature dropped to ambient,
when two more volumes of hexane were added. The product
was isolated by filtration. Further purification (if necessary)
was achieved by heating the solid under reflux with a 1:1
also transferred to a toll manufacturer, but quality suffered,
and subsequent clean up had to be performed before use.2
Purification of Amine 4. As noted above impure samples
were obtained from tollers (assays ranged from 78 to 98%
on a wt/wt basis by HPLC and titration), and these were
cleaned up as follows:
mixture of ethyl acetate and hexane (10 mL total). Mp 162-
1
1
63 °C; H NMR (d
6
-DMSO) δ 10.80 (1H, s), 8.96 (1H,
m), 8.24 (2H, m), 7.94 (1H, d), 4.37 (1H, m), 2.50 (1H, s),
EtOAc (32 kg) was placed in a 30-gal reactor followed
by the impure amine 4 (7.0 kg). The mixture was heated
under reflux for 30 min and then filtered hot through a
Nutsche filter. The procedure was repeated on the filter cake.
Charcoal (Darco, 280 g) was added to the combined filtrates
which was then heated under reflux for 30 min. The mixture
was filtered hot through cartridge filters. The solution was
concentrated to one-third of its original volume by vacuum
distillation and then cooled to ambient temperature. The solid
was collected on a Nutsche filter and allowed to dry under
a stream of dry nitrogen. A second crop was obtained from
the filtrate by heating it to boiling and then setting the jacket
temperature at 60 °C. Hexane (2× the weight of EtOAc
present) was then added. The mixture was cooled to ambient
temperature over 1 h and then filtered through a Nutsche,
drying in a nitrogen stream. Both crops were within
specifications (>98% wt/wt by HPLC and titration, and the
solid was off-white to light yellow in colour).
2
.40-1.80 (4H, m), 1.46 (1H, m), 1.30-1.05 (5H, m), 0.98
13
(3H, d), and 0.82 (3H, t); C (NMR) δ 176.1, 173.7, 171.8,
1
2
41.9, 138.8, 129.5, 125.9, 125.8, 117.6, 52.9, 33.4, 30.2,
8.9, 26.6, 22.1, 17.9, and 13.8; [R] -24.9° (50 mg in 2
D
mL of MeOH)
Alternative Methods. Enzymatic Approach. To a thin
slurry of dimethyl glutamate (25) (8.46 g, 0.04 mol) and
sodium bicarbonate (10.08 g, 0.12 mol) in water was added
(S)-2-methylhexanoyl chloride (11) (5.94 g, 0.04 mol) in THF
(10 mL) over a period of 15 min. The mixture was allowed
to stir at room temperature for 1 h. The THF was evaporated
on a rotary evaporator at room temperature, and the aqueous
layer was extracted with ethyl acetate (30 mL). The organic
layer was washed with aqueous saturated sodium bicarbonate
(30 mL) and then with water (30 mL). The ethyl acetate
layer was dried (MgSO4) and filtered, and the filtrate was
concentrated to an oil. The oil solidified within 5 min. Yield
Preparation of Amine Hydrochloride 17. THF (96 kg)
was charged to a glass-lined 100-gal reactor. The amine 4
10.1 g (88%).
PLE (0.8 mL suspension, from Sigma) was added to a
(12.0 kg) was added, and the mixture was heated to boiling
suspension of dimethyl N-((S)-2-methylhexanoyl)-L-glutamate
24) (7.4 g, 0.26 mol) in 0.01 M phosphate buffer (pH 7) at
and then cooled to ambient temperature. The solution was
then flitered through a sparkler Nutsche filter, returning the
clear mother liquors to the reactor. The solution was cooled
to 0 °C, and then HCl gas was bubbled through at 60 psig
for 15 min. Nitrogen was then bubbled through the mixture
for 15 min. The HCl addition was repeated twice more. After
the final addition, the mixture was stirred for 1 h at 0 °C.
The solid was collected on a Nutsche filter. The reactor, line,
and then cake were washed with THF (11 kg). The solid 17
was dried overnight at ambient temperature on the filter bed
under a stream of dry nitrogen.
Coupling of NCA 16. DMF (40 kg) was added to a 30-
gal reactor followed by the NCA 16 (24 kg). The mixture
was stirred at ambient temperature for 30 min to ensure
dissolution. The pyridine hydrochloride 17 (17.86 kg) was
then added followed by the free amine 4 (1.16 kg). The
reaction was stirred at ambient temperature for 3 days. The
mixture was then filtered, rinsing the reactor and lines with
DMF (10 kg). The filtrate was stirred for a further 2 days.
After this time the free amine concentration should be below
(
room temperature. The pH value was kept at about 7 by the
addition of 1 N sodium hydroxide. After consumption of
0.95 mol equiv of base, the pH was adjusted to 9 by the
addition of 1 N sodium hydroxide, and the mixture was
extracted with ethyl acetate (20 mL). The aqueous layer was
separated and acidified to pH 2-3 by the addition of 1 N
hydrochloric acid and extracted with ethyl acetate (2 × 20
mL). The organic layer was washed with water (20 mL),
4
dried (MgSO ), and filtered, and the filtrate was concentrated
to an oil. The oil solidified on standing. Yield 5.68 g (80%).
Chemical Method to Ester 19. To a cold (0 °C) solution
of 27 (2.73 g, 0.01 mol) in ethyl acetate (20 mL) was added
N-methylmorpholine (1.01 g, 1.1 mL, 0.01 mol), followed
by trimethylacetyl chloride (1.2 g, 1.23 mL, 0.01 mol). After
10 min, the amine 4 (1.19 g, 0.01 mol) was added, and the
mixture was warmed to room temperature. The mixture was
stirred at room temperature for 24 h and then diluted with
10 mL of 1 N HCl. The organic layer was separated, washed
successively with saturated aqueous ammonium chloride
solution (10 mL), saturated sodium bicarbonate solution (10
mL), and water (10 mL), and dried (MgSO ). Filtration and
4
5
% by peak area. It was then used in the subsequent coupling
step as is.
Acid Chloride 11. This procedure had to performed so
concentration of the filtrate gave a pale solid, which was
recrystallised from a mixture of ethyl acetate and hexane
that the acid chloride was ready for the coupling reactions
without any undue delay.
(
35 mL/35 mL) to give a white solid (2.1 g, 54%).
Large-Scale Runs. Many of these procedures were
Thionyl chloride (12.3 L, 20.1 kg) was charged to a 5-gal
reactor under nitrogen and then cooled to 0 °C. 2-Methyl-
hexanoic acid 2 (3.00 kg) was then added, ensuring that
foaming did not occur and the internal temperature did not
exceed 35°. The transfer lines were rinsed with THF (1 L).
The reaction was stirred at 0 °C for 30 min and brought to
limited by the size of equipment available to us. In many
cases, multiple runs had to be performed to obtain sufficient
material. In the case of the NCA 16 this was run at a toll
manufacturer using our procedure. The pyridine amine 4 was
84
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Vol. 8, No. 1, 2004 / Organic Process Research & Development