Efficient synthesis of (S)-2-(cyclopentyloxycarbonyl)-amino-8-nonenoic acid: Key building block for BILN 2061, an HCV NS3 protease inhibitor

Article abstract of DOI:10.1021/op0601924

A new procedure for the practical synthesis of (S)-2-(cyclopen- tyloxycarbonyl)amino-8-nonenoic acid, a key building block for BILN 2061, an HCV NS3 protease inhibitor, has been developed. The key step features a kinetic resolution of racemic 2-acetylamino-8-nonenoic acid with acylase I. In addition, the undesired (R)-2-acetylamino-8-nonenoic acid was recycled after racemization. The procedure was implemented for the production of (S)-2-(cyclopentyloxycarbonyl)amino-8-nonenoic acid on pilot-plant scale.

Full text of DOI:10.1021/op0601924

Organic Process Research & Development 2007, 11, 60−63
Efficient Synthesis of (S)-2-(Cyclopentyloxycarbonyl)-amino-8-nonenoic Acid:
Key Building Block for BILN 2061, an HCV NS3 Protease Inhibitor
Xiao-jun Wang,* Li Zhang, Lana L. Smith-Keenan, Ioannis N. Houpis,§ and Vittorio Farina*
Department of Chemical DeVelopment, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut 06877, U.S.A.
Abstract:
of (Z)-2-acetamido-2,8-nonadienoate using (S,S)-Et-DU-
PHOSRh(COD)OTf,4 followed by the amide-to-carbamate
conversion5 and saponification.3 In this paper, we describe
an alternative synthesis of building block (S)-2-(cyclopen-
tyloxycarbonyl)amino-8-nonenoic acid (3), by kinetic enzy-
matic resolution of the corresponding racemate with acylase
I. This procedure could be reproduced in the pilot plant, after
simple operational adjustments, on a scale up to 100 kg.
A new procedure for the practical synthesis of (S)-2-(cyclopen-
tyloxycarbonyl)amino-8-nonenoic acid, a key building block for
BILN 2061, an HCV NS3 protease inhibitor, has been devel-
oped. The key step features a kinetic resolution of racemic
2-acetylamino-8-nonenoic acid with acylase I. In addition, the
undesired (R)-2-acetylamino-8-nonenoic acid was recycled after
racemization. The procedure was implemented for the produc-
tion of (S)-2-(cyclopentyloxycarbonyl)amino-8-nonenoic acid on
pilot-plant scale.
Results and Discussion
Because R-amino acids constitute one of the most
important classes of natural products exhibiting important
biological functions, a wide variety of synthetic methods are
available for their preparation.6 We focused on the application
of inexpensive acylase I (EC 3.5.1.14) to the L-selective
cleavage of N-acetyl amino acids, according to the extensive
studies by Whitesides.7 Acylase I enzymes isolated from
porcine kidney (PKA) and the fungus Aspergillus sp. (AA)
are both commercially available, inexpensive, stable in
aqueous solution, and possess high specific activity. Con-
sidering the fact that acetylamino acids are good substrates
of acylase I and are readily prepared, we chose 2-acetamido
carboxylic acid (()-9 as our target (Scheme 2) for the
enzymatic resolution. This would allow us to introduce the
cyclopentyloxy group required in BILN 2061 by a simple
acylation of the free amino group of 10 obtained by
enantioselective hydrolysis of (()-9.
7-Chloro-1-heptene (7) was prepared using a known
literature procedure.8 Thus, cross-coupling of 1-1-bromo-4-
chlorobutane (6) with allylmagnesium bromide in the pres-
ence of dilithium tetrachlorocuprate in THF gave 7 in good
yield. Although THF solutions of dilithium tetrachlorocuprate
are commercially available, we prefer its in situ preparation
by simply mixing 2 equiv of anhydrous lithium chloride with
anhydrous copper (II) chloride in THF. This THF solution
of Li2CuCl4 prepared in situ was as effective as the
commercially available one. Alkylation of diethyl acetami-
Introduction
BILN 2061, a new HCV NS3 protease inhibitor, was
recently designed on the basis of a rational approach
involving the use of traditional medicinal chemistry, parallel
synthesis, and structural data for the optimization of binding
and downstream biopharmaceutical properties.1 It has shown
good oral bioavailability and antiviral effect in humans
infected with HCV genotype 1.2 The selection of BILN 2061
as a candidate for further clinical studies necessitated a
practical synthesis suitable for pilot-plant scale.
The structure of BILN 2061 features a 15-membered
macrocyclic tripeptide. In our retrosynthetic approach (Scheme
1), the 15-membered macrocyclic tripeptide can be con-
structed by Ru-catalyzed ring-closing metathesis (RCM) of
an acyclic tripeptide precursor 1,3 followed by etherification
with 2. In turn, the acyclic tripeptide precursor 1 can be
synthesized by fragment condensation of three unnatural
R-amino acids derivatives, 3, 4, and 5.
(S)-2-(tert-Butyloxycarbonyl)amino-8-nonenoic acid had
been prepared on small scale by asymmetric hydrogenation
* Corresponding authors: (X.J.W.) Telephone: (203) 791-6199. Fax: (203)
971-6168. E-mail: xwan@rdg.boehringer-ingelheim.com. (V.F.) Tele-
phone: (203) 791-6625. Fax: (203) 791-6130. E-mail: farinav@
rdg.boehringer-ingelheim.com.
§ Current address: Johnson and Johnson Pharmaceutical R&D, Turnhoutseweg
30, B-2340, Beerse, Belgium.
(1) Llina´s-Brunet, M.; Bailey, M.; Bolger, G.; Brochu, C.; Faucher, A.-M.;
Ferland, J.-M.; Garneau, M.; Ghiro, E.; Gorys, V.; Grand-Maitre, C.;
Halmos, T.; Lapeyre-Paquette, N.; Liard, F.; Poirier, M.; Rheaume, M.;
Tsantritzos, Y.; Lamarre, D. J. Med. Chem. 2004, 47, 1605-1608 and
references therein.
(2) Lamarre, D.; Anderson, P. C.; Bailey, M.; Beaulieu, P.; Bolger, G.; Bonneau,
P.; Boes, M.; Cameron, D. R.; Cartier, M.; Cordingley, M. G.; Faucher,
A.-M.; Goudreau, N.; Kawai, S. H.; Kukolj, G.; Lagace, L.; LaPlante, S.
R.; Narjes, H.; Poupart, M.-A.; Rancourt, J.; Sentjens, R. E.; St. George,
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S. M.; Yong, C.-L.; Llina´s-Brunet, M. Nature 2003, 426 (6963), 186-189.
(3) Faucher, A.-M.; Bailey, M. D.; Beaulieu, P. L.; Brochu, C.; Duceppe, J.-
S.; Ferland, J.-M.; Ghiro, E.; Gorys, V.; Halmos, T.; Kawai, S. H.; Poirier,
M.; Simoneau, B.; Tsantrizos, Y. S.; Llina´s-Brunet, M. Org. Lett. 2004, 6,
2901-2904.
(4) Burk, M. J.; Allen, J. G.; Kiesman, W. F. J. Am. Chem. Soc. 1998, 120,
657-663.
(5) Burk, M. J.; Allen, J. G. J. Org. Chem. 1997, 62, 7054-7057.
(6) (a) Williams, R. M. In Synthesis of Optically ActiVe R-Amino Acids; Baldwin,
J. E., Ed.; Organic Chemistry Series; Pergamon Press: Oxford, 1989. (b)
Maruoka, K.; Ooi, T. Chem. ReV. 2003, 103, 3013-3028. (c) Cativiela, C.;
Diaz-de-Ville`gas, M. D. Tetrahedron: Asymmetry 1998, 9, 3517-3599.
(d) Seebach, D.; Sting, A. R.; Hoffmann, M. Angew. Chem., Int. Ed. Engl.
1996, 35, 2708-2748. (e) Duthaler, R. O. Tetrahedron 1994, 50, 1539-
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(7) (a) Chenault, H. K.; Dahmer, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989,
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1564.
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10.1021/op0601924 CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/20/2006

Scheme 1. Retrosynthetic analysis
Scheme 2. Synthesis of (S)-2-amino-8-nonenoic acid
domalonate with the crude 7 was accomplished in 85% yield
by heating the mixture at 80 °C in DMF for 16 h with a 4:1
mixture of potassium carbonate and cesium carbonate as base
and a catalytic amount of potassium iodide.9 With only
potassium carbonate as base, the alkylation was sluggish,
taking 92 h to reach completion. It is worth mentioning that
the initial attempt to prepare 8 using 7-bromo-1-heptene as
alkylating agent proceeded much more readily than that using
7. However, preparation of 7-bromo-1-heptene by the same
cross coupling of 1,4-dibromobutane with allylmagnesium
bromide gave only a 40% of the monobromide, due to the
formation of 1,9-decadiene, the product of double allylation.
Intermediate 8 can be also used without purification/isolation.
Saponification of the crude 8 was effected by treatment with
potassium hydroxide in aqueous ethanol at 60 °C. Upon
concentration, the aqueous reaction mixture was neutralized
with citric acid to yield (()-9 in 88% yield after recrystal-
lization from isopropanol/water.
°C, the solid product was collected simply by filtration to
provide 10 in g45% yield with >99% ee as determined by
HPLC on chiral support; the reaction was halted when an
assay on the solution indicated 95% ee for enriched (R)-9.
There was no need for a periodic addition of sodium
hydroxide solution to maintain the pH at 7.5-8.0, as
normally required for this type of enzymatic resolution, since
the pH of the reaction mixture showed little change.
Apparently, after L-selective cleavage of the acetamide
bond, the resulting acetic acid partially neutralizes the amino
acid sodium salt formed to the free amino acid 10, which
precipitates out of solution, driving the equilibrium. As a
result, the pH of the solution is self-buffered. The virtually
full precipitation of the desired 10 from the reaction mixture
allowed an exceedingly simple isolation, and the self-
adjusting pH of the reaction mixture obviated the need to
periodically add base to the reaction mixture. These two
features make the process practical, robust, and easy to
perform on large scale.
This racemic material was then subjected to kinetic
resolution with 0.5% (w/w) acylase I in water at pH 7.5-
8.0 in the presence of small amounts of Co(II), an enzyme
cofactor. When the reaction mixture was heated to 37 °C
for 30 min, we noticed the precipitation of the amino acid
10 and little variation of the solution pH.10 After 7 h at 37
As typical of other R-amino acids, the undesired (R)-9
could be recycled through racemization. To illustrate this,
the (R)-enriched 9 sodium salt was fully racemized by heating
in water containing Ac2O.11 After two cycles of racemization,
an overall 71% yield of 10 could be obtained.
We next turned to the preparation of the fragment 3 with
optically pure 10. Treatment of 10 with commercially
(9) (a) Keith, D. D.; Yang, R.; Tortora, J. A.; Weigele, M. J. Org. Chem. 1978,
43, 3713-3716. (b) Rinderknecht, H.; Niemann, C. J. Am. Chem. Soc. 1951,
73, 4259-4260.
(10) In a few cases, the precipitation of (S)-15 was delayed, leading to a drop in
pH. It rose once precipitation began.
(11) Johnson, E. P.; Chen, G.-P.; Fales, K. R.; Lenk, B. E.; Szendroi, R. J.;
Wang, X.-J.; Carlson, J. A. J. Org. Chem. 1995, 60, 6595-6598.
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Scheme 3. Completion of the synthesis
available cyclopentyl chloroformate under Schotten-Bau-
mann conditions gave 3 in quantitative yield. The crude, oily
material may be used directly for the formation of the
tripeptide precursor 1. However, for more convenient
handling and long-term storage of this material, the fragment
3 was isolated as the highly crystalline dicyclohexylamine
salt 11 in 85% yield by a simple treatment of the crude 3
with dicyclohexylamine in methyl tert-butyl ether (MTBE,
Scheme 3).
and 3% NaCl (6.0 L) and was distilled (20 °C, 100 mmHg)
to a low volume. Assay by 1H NMR indicated 450 g of 7 in
the residue (83%). This crude material, which contained 50%
w/w THF admixed with 7, was taken to the next step without
further purification. The physical data (NMR, IR) obtained
on this product were identical to those reported in the
literature.12
Diethyl (Acetamido)(6-hepten-1-yl)propanedioate (8).
Potassium iodide (305 g, 1.84 mol), potassium carbonate (635
g, 4.60 mol), and cesium carbonate (1.50 kg, 4.60 mol) were
added to a mixture of crude 7 (prepared as above, pooled
from three runs, 1.23 kg estimated by NMR assay, 9.20 mol)
and diethyl acetamidomalonate (1.95 kg, 9.20 mol) in
anhydrous DMF (8.0 L). The reaction mixture was stirred
at 80 °C for 16 h, then was cooled to RT, and water (8.0 L)
was added, followed by EtOAc (16 L). The organic layer
was washed with water (2 × 8.0 L), and the organic solvent
was distilled off. The residue, containing some residual ethyl
acetate, was assayed by 1H NMR, which showed 2.45 kg of
8 (85%). This crude material was used in the next step
without further purification. A small aliquot was purified by
flash column chromatography to give 8 as a colorless oil:
1H NMR (400 MHz, CDCl3) δ 1.10 (m, 2H), 1.25 (t, J )
7.2 Hz, 6 H), 1.33 (m, 4H), 2.02 (m, 2 H), 2.03 (s, 3H),
2.32 (dt, J ) 2.4 and 6.0 Hz, 2H), 4.23 (q, J ) 7.2 Hz, 4
H), 4.95 (dd, J ) 1.6 and 15.6 Hz, 2H), 5.77 (m, 1H), 6.76
(s, 1H); 13C NMR (400 MHz, CDCl3) δ 168.9, 168.2, 138.8,
114.4, 66.6, 62.4, 33.6, 32.0, 28.7, 28.6, 23.4, 23.0, 14.0;
HRMS (APCI) calcd for C16H29NO5 314.1961, found
314.1976.
(()-2-Acetamido-8-nonenoic acid (9). To a solution of
diethyl (acetamido)(6-hepten-1-yl)propanedioate (8, 1.89 kg,
6.02 mol) in ethanol (10 L) and water (1.0 L) was added
potassium hydroxide (1.01 kg, 18.1 mol) at RT. The solution
was stirred at 60 °C for 16 h. The solution was partially
evaporated (to ∼3 L), and diluted with water (6.0 L). The
aqueous phase was washed with ethyl acetate (2 × 1.3 L),
and the pH was adjusted to 4-5 by addition of citric acid
(∼1.3 kg, 6.76 mol). The mixture was stirred at 100 °C for
6 h and then cooled to RT. The pH was further adjusted to
3.5 by adding 3 N HCl. The aqueous layer was extracted
with ethyl acetate (2 × 3.0 L). Evaporation of the solvents
was followed by recrystallization of the residue from
isopropanol/water to give 9 (1.13 kg, 88%) as a white solid:
Conclusion
In summary, we have developed a practical procedure for
preparation of (S)-2-(cyclopentyloxycarbonyl)amino-8-non-
enoic acid (3) with >99% ee in 28% overall yield over six
steps. A recycling process was established for the kinetic
resolution to permit more complete conversion of the material
to the desired L-amino acid. This can boost the overall yield
to g40%. The above synthesis of building block 11 was
scaled, with minor operational adjustments, to >100 kg, as
part of the manufacturing of BILN 2061 for clinical
development.13
Experimental Section
General. Acylase I was purchased from Sigma and stored
at 0 °C as instructed. All other reagents and solvents were
purchased from commercial sources and used without further
drying and purification. NMR spectra of all compounds were
recorded on a Bruker-Biospin DPX 400 NMR spectrometer.
Enantiomeric excess determinations were carried out on a
Chiralpak AD-RH column (0.46 cm × 15 cm) using 0.02%
phosphoric acid in water and acetonitrile as eluent (65:35),
using a flow rate of 0.7 mL/min at a column temperature of
35 °C and a detection wavelength of 205 nm. The analyte
concentration was typically 1-2 mg/mL in water/acetonitrile
(1:1). NMR assays were carried out by adding a weighed
amount of ethylene carbonate to a determined volume of the
solution to be analyzed (in CDCl3), and quantitating the
analyte by NMR integration vs the known concentration of
internal standard.
1-Chlorohept-6-ene (7). To a solution of LiCl (69.0 g,
1.63 mol) in THF (7.0 L) was added and CuCl2 (109 g, 0.821
mol), and the mixture was stirred at 20 °C for 1 h under N2.
The solution was then cooled to 0 °C, and 1-bromo-4-
chlorobutane (6, 700 g, 4.08 mol) was added. A solution of
1 M allylmagnesium bromide (5.50 L, 5.50 mol) in THF
was added over a period of 30 min at 0 °C. The mixture
was stirred at this temperature for 2 h and then quenched
with 10% H2SO4 (5.0 L) followed by addition of MTBE (9.0
L). The organic layer was washed with water (2 × 6.0 L)
(12) Perrine, T. D. J. Org. Chem. 1953, 18, 1356-1367.
(13) Yee, N. K.; Farina, V.; Houpis, I. N.; Haddad, N. Frutos, R. P.; Gallou, F.;
Wang, X.-J.; Wei, X.; Simpson, R. D.; Feng, X.; Fuchs, V.; Xu, Y.; Tan,
J.; Zhang, L.; Xu, J.; Smith-Keenan, L. L.; Vitous, J.; Ridges, M. D.; Spinelli,
E. M.; Johnson, M.; Donsbach, K.; Nicola, T.; Brenner, M.; Winter, E.;
Kreye, P.; Samstag, W. J. Org. Chem. 2006, 71, 7133-7145.
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1
mp 129-131 °C. H NMR (400 MHz, CDCl3) δ 1.36 (m,
diluted with water (3 L). The aqueous layer was washed with
ethyl acetate (1.5 L); 1 N HCl was added to the solution to
bring the pH to 3. The mixture was then extracted with tert-
butyl methyl ether (4 L). Dicyclohexylamine (275 g, 1.50
mol) was added to the extracts over a period of 30 min at
50 °C. The suspension was cooled to room temperature and
stirred for an additional 4 h. The slurry was filtered, and the
filter cake was washed with methyl tert-butyl methyl ether
(0.5 L). The solid was dried to give dicyclohexylamine salt
11 (530 g, 85%) as a white solid: mp 90-93 °C. 1H NMR
(400 MHz, DMSO-d6) δ 1.10 (m, 2H), 1.25 (m, 14 H), 1.60
(m, 10 H), 1.69 (m, 8H), 2.02 (m, 6 H), 3.64 (t, J ) 6 Hz,
1H), 4.96 (m, 3H), 5.76 (m, 1H), 6.22 (d, J ) 6.8 Hz, 1 H);
13C NMR (400 MHz, DMSO-d6) δ 174.3, 155.2, 138.7,
114.5, 75.6, 55.0, 51.6, 33.1, 32.4, 32.3, 29.2, 28.5, 28.3,
25.1, 24.8, 24.2, 23.2; MS (CI): m/z 284 (M+ + 1); Anal.
Calcd for C27H48N2O4: C, 69.79; H, 10.41; N, 6.03. Found:
C, 69.52; H, 10.49; N, 5.80.
Racemization of (R)-2-Acetamido-8-nonenoic Acid
[(R)-9]. To a solution of the sodium salt of (R)-2-acetamido-
8-nonenoic acid [(R)-(9)] (850.0 g, 3.36 mol) in water (8 L)
was added acetic anhydride (1.5 L, 16.20 mol) at 60 °C over
a period of 6 h with stirring under nitrogen. The reaction
mixture was stirred at 60 °C for an additional 16 h and cooled
to RT. Concentrated HCl was added to bring the pH to about
3. The mixture was extracted with ethyl acetate (2 × 3 L).
The combined extracts were washed with water (1 L) and
concentrated to dryness. The NMR weight % assay and
HPLC analysis on chiral support indicated 847 g of racemic
9 in the organic residue (99%). This crude material was
directly taken to the next cycle of resolution without
purification.
6H), 1.72 (m, 1 H), 1.88 (m, 1H), 2.03 (m, 2 H), 2.06 (s,
3H), 4.60 (dt, J ) 5.6, 7.6 Hz, 1H), 4.97 (dd, J ) 1.6, 15.6
Hz, 2H), 5.80 (m, 1H), 6.20 (d, J ) 8 Hz, 1H); 13C NMR
(400 MHz, CDCl3) δ 175.5, 171.5, 138.8, 114.4, 52.5, 33.6,
32.0, 28.7, 28.6, 25.0, 22.8; MS (CI): m/z 214 (M+ + 1);
Anal. Calcd for C11H19NO3: C, 61.95; H, 8.98; N, 6.57.
Found: C, 62.24; H, 9.17; N, 6.52.
(S)-2-Amino-8-nonenoic Acid (10). To a slurry of
racemic 9 (670 g, 3.14 mol) in water (7.0 L) were added
sodium hydroxide (125 g, 31.2 mol) and CoCl2‚6H2O (500
mg, 2.10 mmol) at RT to form a solution. The pH was
adjusted to 7.8 by addition of 0.1 N NaOH. The solution
was heated to 38.5 °C under stirring, and acylase I (0.7 g)
was added. The reaction mixture was stirred at 38.5 °C for
7 h. After adjusting the pH to 7.6 using acetic acid (if
needed), the slurry was cooled to RT and filtered. The
resulting filter cake was washed with water (1.0 L), and a
10:1 mixture of water/methanol (0.75 L). The solid was dried
to give 10 (230 g, 45%) as an off-white solid: mp 200-
201 °C dec. HPLC on chiral support indicated >99% ee. 1H
NMR (400 MHz, NaOD/D2O) δ 1.28 (m, 4H), 1.36 (m, 2
H), 1.49 (m, 1 H), 1.55 (m, 1H), 2.02 (m, 2 H), 3.17 (t, J )
6 Hz, 1H), 4.97 (dd, J ) 10, 18 Hz, 2H), 5.61 (m, 1H); 13
C
NMR (NaOD/D2O) δ 183.0, 139.4, 113.1, 55.2, 33.7, 32.0,
27.3, 27.0, 23.8. MS (CI): m/z 172 (M+ + 1); Anal. Calcd
for C9H17NO2: C, 63.13; H, 10.01; N, 8.18. Found: C, 62.79;
10.11; N, 7.90.
Dicyclohexylamine Salt of (S)-2-(Cyclopentyloxycar-
bonyl)amino-8-nonenoic Acid (11). To a stirred slurry of
10 (228 g, 1.33 mol) in a 1:1 mixture of THF/water (4 L)
was added sodium hydroxide (53 g, 1.33 mol) to form a
solution. Sodium carbonate (142 g, 1.33 mol) was added,
followed by addition of cyclopentyl chloroformate (230 g,
1.47 mol) at RT over a period of 35 min. The reaction
mixture was stirred at RT for an additional 30 min and
Received for review September 20, 2006.
OP0601924
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