Practical Synthesis of BILA 2157 BS, a Potent and Orally Active Renin Inhibitor: Use of an Enzyme-Catalyzed Hydrolysis for the Preparation of Homochiral Succinic Acid Derivatives

Article abstract of DOI:10.1021/jo990321x

We have developed a highly convergent and stereoselective synthesis of BILA 2157 BS, a potent and orally active renin inhibitor. The synthesis proceeds in 15 distinct chemical steps (with several integrated, multistep operations) from aminodiol 4. The key step in the synthesis involves the use of an enantiospecific, enzyme-catalyzed hydrolysis of a substituted succinate diester to provide a homochiral succinic acid derivative in 98% enantiomeric excess (≥2.5 kg scale). Recycling of the unwanted enantiomer is accomplished through base-catalyzed racemization, leading to an efficient deracemization of the starting racemic diester. The entire sequence proceeds without chromatographic purifications and delivers the product with >97% homogeneity. In addition, compared to the previously reported syntheses of BILA 2157 BS, this approach avoids the use of expensive chiral auxiliaries and cryogenics and, thus, should be amenable to the preparation of large quantities of this peptidomimetic inhibitor.

Full text of DOI:10.1021/jo990321x

6622
J . Org. Chem. 1999, 64, 6622-6634
P r a ctica l Syn th esis of BILA 2157 BS, a P oten t a n d Or a lly Active
Ren in In h ibitor : Use of a n En zym e-Ca ta lyzed Hyd r olysis for th e
P r ep a r a tion of Hom och ir a l Su ccin ic Acid Der iva tives
Pierre L. Beaulieu,* J ames Gillard,* Murray Bailey, Christian Beaulieu,
J ean-Simon Duceppe, Pierre Lavalle´e, and Dominik Wernic
Boehringer Ingelheim (Canada) Ltd., Bio-Me´ga Research Division, 2100 Cunard Street, Laval,
Que´bec, Canada, H7S 2G5
Received February 19, 1999
We have developed a highly convergent and stereoselective synthesis of BILA 2157 BS, a potent
and orally active renin inhibitor. The synthesis proceeds in 15 distinct chemical steps (with several
integrated, multistep operations) from aminodiol 4. The key step in the synthesis involves the use
of an enantiospecific, enzyme-catalyzed hydrolysis of a substituted succinate diester to provide a
homochiral succinic acid derivative in 98% enantiomeric excess (g2.5 kg scale). Recycling of the
unwanted enantiomer is accomplished through base-catalyzed racemization, leading to an efficient
deracemization of the starting racemic diester. The entire sequence proceeds without chromato-
graphic purifications and delivers the product with >97% homogeneity. In addition, compared to
the previously reported syntheses of BILA 2157 BS, this approach avoids the use of expensive
chiral auxiliaries and cryogenics and, thus, should be amenable to the preparation of large quantities
of this peptidomimetic inhibitor.
In tr od u ction
The renin-angiotensin system (RAS) plays a central
role in the regulation of blood pressure and, over the
years, has been fruitful ground for the search for new
therapies for the treatment of hypertension and conges-
tive heart failure.1,2 Following the advent and initial
success of angiotensin converting enzyme (ACE) inhibi-
tors, the occurrence of side effects associated with their
use3 has stimulated further research to identify other
sites for therapeutic intervention along the RAS.4 Incen-
tive for the development of novel therapies based on
inhibition of the aspartyl protease renin,5 the first
implicated enzyme along this pathway, is derived from
the fact that renin, unlike ACE, acts on a single known
substrate (angiotensinogen). Interference with this event
is thus less likely to result in unwanted side effects.
Using the sequence of angiotensinogen as a starting
point for rational design, replacement of the cleavable
Leu-Val amide bond of this substrate by nonhydrolyzable
isosteres quickly led to the discovery of potent and
specific inhibitors of renin.1,2 However, despite extensive
efforts directed at the search for small, orally active
inhibitors of the enzyme, lead compounds have generally
remained large (MW > 600), peptidomimetic-like mol-
ecules, lacking adequate physicochemical properties for
oral absorption.
F igu r e 1.
Our own efforts in this field culminated in the discov-
ery of BILA 2157 BS (1), a potent and specific inhibitor
of human renin (Figure 1).6 Despite a high molecular
weight (MW 727) and significant amide character, BILA
2157 BS exhibited an encouraging pharmacological pro-
file. Oral bioavailability of 1 in cynomolgus monkey was
40%, and the compound elicited a statistically significant
lowering of blood pressure in sodium-depleted animals
at a dose of 3 mg/kg.6 These encouraging results led to
* To whom correspondence should be addressed. E-mail: pbeaulieu@
lav.boehringer-ingelheim.com; jgillard@lav.boehringer-ingelheim.com
(1) Greenlee, W. J . Med. Res. Rev. 1990, 10, 173.
(2) Rosenberg, S. H. Renin Inhibitors. In Progress in Medicinal
Chemistry; Ellis, G. P., Luscombe, D. K., Eds.; Elsevier: New York,
1995; Vol. 32, p 32.
(3) Side effects encountered with the use of ACE inhibitors include
cough (6-14% of patients), skin rash, and angioneurotic oedema.1,2
(4) Most notable are nonpeptide AII receptor antagonists such as
losartan; see, for example: (a) Buchholz, R. A.; Lefker, B. A.; Ravi
Kiron, M. A. Ann. Rep. Med. Chem. 1993, 28, 69. (b) Greenlee, W. J .;
Siegl, P. K. S. Ann. Rep. Med. Chem. 1992, 27, 59.
(6) Simoneau, B.; Lavalle´e, P.; Anderson, P. C.; Bailey, M.; Bantle,
G.; Berthiaume, S.; Chabot, C.; Fazal, G.; Halmos, T.; Ogilvie, W.;
Poupart, M.-A.; Thavonekham, B.; Xin, Z.; Thibeault, D.; Bolger, G.;
Panzenbeck, M.; Winquist, R.; J ung, G. L. Bioorg. Med. Chem. 1999,
7, 489.
(5) Boger, J . Ann. Rep. Med. Chem. 1985, 20, 257.
10.1021/jo990321x CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/11/1999

Synthesis of BILA 2157 BS
J . Org. Chem., Vol. 64, No. 18, 1999 6623
Sch em e 1
the selection of BILA 2157 BS as a candidate for further
preclinical evaluation. For these studies, large quantities
of the inhibitor became necessary, requiring the develop-
ment of a practical synthesis. In this account, we describe
a stereoselective and convergent synthesis of 1, which
makes use of an environmentally sound and economical
enzymatic hydrolysis for the preparation of a key homo-
chiral succinic derivative.
sembly depicted in Scheme 1. Logical disconnections are
provided by the amide linkages on both sides of the
succinyl core, leading to three key intermediates 2-4.6
This scheme was subsequently modified to use vinyl
bromide 7 (the vinyl bromide being a precursor to the
aminothiazolyl heterocycle),9 instead of 3, to avoid partial
epimerization of the P2 position previously encountered
during coupling with the aminodiol 4.10 Contamination
of the final product, resulting from epimers at P2, was
found to be exceedingly difficult to eliminate in the final
stages of the synthesis. The preparation of 1 thus
proceeded via crystalline bromide 5, followed by con-
struction of the heterocycle onto the complete inhibitor
backbone.10
Resu lts a n d Discu ssion
The large-scale synthesis of peptidomimetic inhibitors,
in general, represents a major synthetic challenge to the
organic chemist.7 In the case of renin inhibitors, the high
cost usually associated with synthetic complexity must
be minimized to allow possible competition with ACE
inhibitors (now entering the generic market) and the
more recently introduced nonpeptide angiotensin II
antagonists.4 Furthermore, due to the chronic nature and
widespread occurrence of high blood pressure related
conditions, lifetime treatment costs must be maintained
at acceptable levels. These factors are important con-
straints that must be taken into consideration in the
planning of a synthetic strategy.
BILA 2157 BS has four asymmetric centers, three of
which are part of its dihydroxyethylene transition-state
mimic (P1-P1′).8 The left-hand side (P4-P3) is a structur-
ally simple, achiral tertiary amide derivative. The re-
maining chiral center is contained within the central
succinyl core of the molecule and carries a basic ami-
nothiazolyl P2 side chain. This heterocycle plays a critical
role in the pharmacokinetic profile of 1.6
Because of the amorphous nature of BILA 2157 BS,11
and despite numerous attempts to prepare crystalline
addition salts, final purification of 1 was accomplished
by flash chromatography.
Secon d -Gen er a tion Syn th esis. Following our first
scale-up campaign of BILA 2157 BS,10 we envisaged
requirements for multikilogram quantities of the inhibi-
tor, in support of full toxicology studies. With this goal
in mind, the original route to 1 suffered from several
limitations that severely restricted its application to
large-scale preparations. Most problematic were the
syntheses of succinyl fragment 7 and aminodiol 4. The
former required the use of an expensive chiral auxiliary,
three cryogenic steps, and the use of pyrophoric and
costly alkyllithium reagents. The reported cyanohydrin
route to aminodiol 412 was found to be capricious and
subject to low overall yields, in addition to requiring the
use of large amounts of expensive and highly toxic
F ir st-Gen er a tion Syn th esis. The “medicinal chem-
istry” route to BILA 2157 BS, used during SAR studies
leading to its discovery, is based on a convergent as-
(9) (a) Morton, H. E.; Leanna, M. R. Tetrahedron Lett. 1993, 34,
4481. (b) Leanna, M. R.; Morton, H. E. Tetrahedron Lett. 1993, 34,
4485.
(7) Beaulieu, P. L.; Lavalle´e, P.; Abraham, A.; Anderson, P. C.;
Boucher, C.; Bousquet, Y.; Duceppe, J .-S.; Gillard, J .; Gorys, V.; Grand-
Maˆıtre, C.; Grenier, L.; Guindon, Y.; Guse, I.; Plamondon, L.; Soucy,
F.; Valois, S.; Wernic, D.; Yoakim, C. J . Org. Chem. 1997, 62, 3440.
(8) Luly, J . R.; BaMaung, N.; Soderquist, J .; Fung, A. K. L.; Stein,
H.; Kleinert, H. D.; Marcotte, P. A.; Egan, D. A.; Bopp, B.; Merits, I.;
Bolis, G.; Greer, J .; Perun, T. J .; Plattner, J . J . J . Med. Chem. 1988,
31, 2264.
(10) Simoneau, B.; Lavalle´e, P.; Bailey, M.; Duceppe, J .-S.; Grand-
Maˆıtre, C.; Grenier, L.; Ogilvie, W. W.; Poupart, M.-A.; Thavonekham,
B. Can. J . Chem., in press.
(11) BILA 2157 BS exists in solution as
a mixture of several
rotamers, due to the presence of two tertiary amide functionalities.
Coupled to a high peptidic content, this phenomena confers amorphous
physical properties to the molecules which have not allowed purifica-
tion of the inhibitor by crystallization.

6624 J . Org. Chem., Vol. 64, No. 18, 1999
Beaulieu et al.
Sch em e 2
Sch em e 3
Sch em e 4
trimethylsilylcyanide. The dihydroxylation route to 413
appeared more practical on a large scale, but suffered
from modest control of diastereoselectivity during intro-
duction of the hydroxyl groups. Finally, an alternative
to chromatographic purification of the final product was
required for large-scale synthetic purposes.
The synthesis of the left-hand side (2, Scheme 2) of
BILA 2157 BS, used in our initial scale-up, was judged
suitable for large-scale preparations. No attempts were
made to further improve this procedure, which delivers
this fragment in 60% overall yield (kg scale) from
commercially available cyclohexylmethylamine 9 and
2-(2-methylaminoethyl)pyridine 10, without need for
purifications. The synthesis of 2 has been described
elsewhere.10
P r ep a r a tion of Ra cem ic Su ccin yl F r a gm en ts.
After consideration of several possibilities, we settled on
preparing the succinyl core (14) in racemic form, followed
by separation of the two enantiomers by resolution. This
strategy avoids the use of an expensive chiral auxiliary
and seemed a particularly attractive methodology since
it had the obvious possibility to be coupled with recycling
of the unwanted antipode through racemization (a pro-
cess often referred to as deracemization).14
While classical resolution processes employing diaster-
eomeric salt formation are widely used on a large scale,
and often provide the most economical way of preparing
enantiomerically pure compounds,7,14 we opted to inves-
tigate the use of enzymes for the stereospecific hydrolysis
protocol illustrated in Scheme 3. Factors influencing our
decision included the following: lower cost associated
with such a catalytic process, the use of water as solvent,
minimal waste management, operational simplicity, and
availability/low cost of reagents. The use of an esterase
enzyme to carry out the stereospecific hydrolysis of
racemate (R,S)-14, coupled to recycling of (R)-14 through
racemization, seemed most efficient and suited to our
application.15 The racemic diester (R,S)-15 (Scheme 4)
was selected as the substrate for this enzymatic resolu-
tion protocol. The â-tert-butyl ester functionality, we
believed, would remain unaffected throughout the process
and ensure a good control of chemoselectivity between
the two carboxylic acid functions during the hydrolysis
step. We also chose the more readily available and less
costly vinylic chloride derivative (X ) Cl in Scheme 3),
rather then the previously used bromide, as precursor
to the aminothiazolyl heterocycle.10 The preparation of
racemic (R,S)-15 shown in Scheme 4 is based on classical
malonate alkylation chemistry. Dimethylmalonate was
first alkylated with 2,3-dichloropropene 16 using sodium
methoxide as a base. A large excess (4 equiv) of the
malonate was used to minimize formation of dialkylated
products, but unreacted material was recovered by simple
distillation and continuously recycled (small amounts of
the desired monoalkylated product 17 co-distill with
dimethylmalonate and recycling of the distillate allows
partial recovery and an increase in yield of the product).
In this way, alkylated malonate 17 was isolated in 70%
yield after purification by vacuum distillation (three
alkylation cycles) and was contaminated with <3% of
dialkylated material. A second alkylation was then
(12) (a) Luly, J . R.; Hsiao, C.-N.; BaMaung, N.; Plattner, J . J . J .
Org. Chem. 1988, 53, 6109. (b) Rosenberg, S. H.; Spina, K. P.; Woods,
K. W.; Polakowski, J .; Martin, D. L.; Yao, Z.; Stein, H. H.; Cohen, J .;
Barlow, J . L.; Egan, D. A.; Tricarico, A.; Baker, W. R.; Kleinert, H. D.
J . Med. Chem. 1993, 36, 449. For other approaches to the synthesis of
4, including scale-up of
a modified cyanohydrin route, see: (c)
Schwindt, M. A.; Belmont, D. T.; Carlson, M.; Franklin, L. C.;
Hendrickson, V. S.; Karrick, G. L.; Poe, R. W.; Sobieray, D. M.; Van
De Vusse, J . J . Org. Chem. 1996, 61, 9564 and references therein.
(13) Krysan, D. J .; Rockway, T. W.; Haight, A. R. Tetrahedron:
Asymmetry 1994, 5, 625.
(14) (a) Collins, A. N.; Sheldrake, G. N.; Crosby, J . Chirality in
industry. The commercial manufacture and applications of optically
active compounds; J ohn Wiley: New York, 1992. (b) Collins, A. N.;
Sheldrake, G. N.; Crosby, J . Chirality in industry II. Developments in
the manufacture and applications of optically active compounds; J ohn
Wiley: New York, 1997.
(15) (a) Bailey, M. D. U.S. Patent 5 808 085, 1998. (b) Bailey, M.;
Adamson, D.; Halmos, T.; Grand-Maˆıtre, C. Tetrahedron: Asymmetry
1999, in press. Similar enzymatic hydrolysis protocols for succinate
derivatives have been reported by other groups: (c) Wirz, B.; Soukup,
M. Tetrahedron: Asymmetry 1997, 8, 187. (d) Ozaki, E.; Sakashita, K.
Chem. Lett. 1997, 741. (e) Guibe´-J ampel, E.; Rousseau, G.; Salau¨n, J .
J . Chem. Soc., Chem. Commun. 1987, 1080.

Synthesis of BILA 2157 BS
J . Org. Chem., Vol. 64, No. 18, 1999 6625
Sch em e 5
performed on 17 using tert-butyl bromoacetate to provide
crude triester 18 in quantitative yield. This alkylation,
originally carried out using strong base (KO-t-Bu or
LiHMDS) and anhydrous conditions, was modified and
ultimately performed using solvent-free phase-transfer
conditions.16 The reaction took place with 10 N NaOH in
the presence of a catalytic amount of benzyltriethylam-
monium chloride, both starting materials, and the gener-
ated product serving as the organic medium. It is
noteworthy that, despite the use of 10 N NaOH, no
hydrolysis of the methyl esters occurred, and crude 18
was obtained in quantitative yield by simple phase
separation. The resistance of either 17 or 18 to saponi-
fication under the strongly basic reaction conditions is
rationalized in terms of the high rate of alkylation of the
anion derived from 17 and the negligible solubility of 18
in the aqueous phase. In an attempt to use less expensive
tert-butyl chloroacetate as the electrophile, the rate of
alkylation was reduced sufficiently for hydrolysis of the
methyl esters to take place, and none of 18 was isolated.
The absence of solvents in this reaction minimizes the
generation of wastes and impact on the environment.
Finally, decarboxylation of 18 to produce racemic (R,S)-
15 was accomplished by initial monosaponification of one
methyl ester using KOH in a homogeneous methanolic
solution, followed by acidification, extraction of the
monoacid into toluene, and refluxing of the extract to
induce decarboxylation. (R,S)-15 was isolated by vacuum
distillation in 80% yield overall from 17 (g2.5 kg scale).
En a n tiosp ecific, En zym a tic Hyd r olysis of Ra ce-
m ic Su ccin a tes. Having secured access to large quanti-
ties of racemic (R,S)-15, we then addressed the key
enzyme-catalyzed hydrolysis step. The use of enzymes
for the stereospecific hydrolysis of esters is a well-
established, highly practical process for the large-scale
synthesis of optically active substances.15,17
After several esterase enzymes were screened for their
effectiveness in the enantiospecific hydrolysis described
in Scheme 3, a crude, readily available, and inexpensive
serine protease (Subtilisin Carlsberg) preparation,
Alcalase,18a was identified as the most promising candi-
date for our purpose. Following optimization of several
reaction parameters (solvents, temperature, time, pH),15a,b
it was found that hydrolysis of the (S)-enantiomer of
(R,S)-15 using a catalytic amount of a crude (food grade)
Alcalase 2.4L preparation18a proceeded smoothly at pH
7.5-8.5 to give (S)-19 in 48% yield and 98% ee as
determined by HPLC analysis on a chiral support (see
figure in the Supporting Information and Experimental
Section for details). The use of purified Subtilisin Carls-
berg did not offer any advantages over the crude prepa-
ration. Unreacted ester (R)-15 (51% yield, 94% ee) and
acid (S)-19 were conveniently separated using a simple
acid-base extraction protocol. The hydrolysis was carried
out on g2.5 kg of racemate, and the crude products could
be used without need of purification. The absolute
stereochemistry of isolated acid (S)-19 was unambigu-
ously determined with the help of an X-ray crystal
structure of a more advanced intermediate 31 (vide infra).
Recycling of the unwanted (R)-15 ester through racem-
ization was accomplished by heating neat (80 °C) with a
catalytic amount of DBU (previously carried out using
stoichiometric amounts of DBU in refluxing toluene).15b
Following three resolution/racemization cycles, the over-
all yield of (S)-19 (98% ee) was increased to 82%,
resulting in efficient deracemization of (R,S)-15.
We wondered if the enzymatic protocol described above
could be carried out on a more advanced intermediate
encompassing the P4-P2 portion of the inhibitor. This
strategy appears less efficient at first glance: large-scale
chemistry dictates that the most economical route is
usually one that carries the least amount of material
through the sequence; therefore, resolution processes
should be implemented as early as possible in a synthetic
scheme, unless the unwanted isomer is recyclable. This
approach however, would advantageously delay introduc-
tion of aminodiol 4 to a latter stage of the synthesis, thus
minimizing requirements of this more valuable fragment.
We therefore examined the enzymatic hydrolysis of
succinate derivative (R,S)-21 as illustrated in Scheme 6.
Cleavage of the tert-butyl ester of (R,S)-15 with 4 N
HCl in dioxane gave crystalline acid (R,S)-20 (85% yield),
which was coupled to P4-P3 (2) using a pivaloyl mixed
anhydride. Crude racemate (R,S)-21 was obtained as an
oil in quantitative yield. Without purification, this crude
ester was submitted to the enzymatic hydrolysis protocol
under conditions identical to those used for (R,S)-15.
Crude ester (R)-21 was isolated in 52% yield and 97.6%
ee as shown by HPLC analysis on a chiral support (see
the Experimental Section for details). Acid (S)-22 (94%
ee), on the other hand, was isolated in lower yield (37-
38%), presumably a consequence of its increased water
solubility. Both compounds were obtained as oils but were
of sufficient purity to be used without purification. Ester
(R)-21 was easily recycled by refluxing with catalytic
NaOMe in methanol, again demonstrating potential for
deracemization (Scheme 6). The absolute stereochemical
assignment for (S)-22 is based on its convergence to a
more advanced intermediate for which stereocenters had
been assigned with the help of a crystal structure (vide
infra).
(16) Singh, R. K. Synthesis 1985, 54.
(17) (a) Enzymes in synthetic organic chemistry; Wong, C. H.,
Whitesides, G. M., Eds.; Elsevier Science, Inc.: New York, 1994. (b)
Enzyme catalysis in organic synthesis. A comprehensive handbook;
Drauz, K., Waldmann, H., Eds.; VCH Publishers: New York, 1995;
Vol. 1.
(18) (a) Alcalase is a crude enzyme preparation whose main com-
ponent (Subtilisin A, Subtilisin Carlsberg) is a serine endopeptidase.
The crude Alcalase 2.4 L enzyme preparation is available in bulk from
Novo Nordisk Biochem North America, Inc., Franklinton, NC 27525.
(b) Since the extent of conversion for these reactions was not accurately
measured, only approximate E values can be determined for these two
substrates; see: Chen, C.-H.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J .
J . Am. Chem. Soc. 1982, 104, 7294.
Both resolution schemes (Schemes 5 and 6) attest to
the versatility and scope of the Alcalase-mediated hy-
drolysis. The enzyme indiscriminately carries out an
almost stereospecific hydrolysis of a simple, lipophilic

6626 J . Org. Chem., Vol. 64, No. 18, 1999
Beaulieu et al.
Sch em e 6
Sch em e 7
Sch em e 8
diester 15 (E > 200)18b or a more complex amide deriva-
tive 21 (E > 100).18b This observation is consistent with
reports of high levels of tolerance at the P2 and P3
positions (and beyond) of substrates in protease-catalyzed
reactions (including subtilisin).17a This procedure for the
preparation of homochiral succinic acid derivatives should
find wide application due to its simplicity, versatility,
efficiency, availability of the enzyme, and practicality for
large scale production.
Ela bor a tion of th e Am in od iol F u n ction a lity. Sev-
eral reports have appeared in the literature that describe
syntheses of transition-state isostere 4.8,12,13 Two routes
are particularly noteworthy as they have been shown to
be amenable to scale-up. Both make use of protected (S)-
cyclohexyalaninal 23 as chiral starting material (Scheme
7). The first route12 proceeds via cyanohydrin 24, which
is then elaborated in a series of steps into aminodiol 4.
The second strategy converts aldehyde 23 into alkene 25
via a Wittig condensation.13 Dihydroxylation of the alkene
then gives a 4:1 mixture of diols in favor of the desired
relative stereochemistry present in 4. While both routes
proceed with comparable efficiencies (31-36% yield
overall), the dihydroxylation approach requires less
chemical operations and thus seemed more attractive to
us.
carbamate protecting group of 25 by a more complex and
chiral entity, such as the P2 portion of our inhibitor,
would favorably influence facial diastereoselectivity in
the dihydroxylation of the double bond. To this end, three
olefinic substrates were prepared as depicted in Scheme
8. Starting with resolved acid (S)-19, coupling with
readily available allylic amine hydrochloride 2613 gave
ester 27. Deprotection of the carboxyl group under strong
acid conditions gave 28, which was coupled to P4-P3 (2)
to give 29, incorporating the complete backbone of BILA
2157 BS.
Dihydroxylation of compounds 27-29, unlike carbam-
ate 25, is potentially complicated by the presence of the
vinylic chloride functionality at P2, which in principle is
also susceptible to electrophilic attack by oxidizing
agents. Using the oxidation conditions previously re-
ported in the literature for alkene 25 (2.5 mol % OsO4/
2.5 equiv of N-methylmorpholine N-oxide/2-propanol)13
we found that substantial oxidation of the vinylic chloride
was taking place, leading to complex mixtures of prod-
ucts. Careful study of dihydroxylation parameters (sol-
vent, water content, co-oxidant, temperature, additives)
led to the optimized conditions shown in Table 1, which
were then applied to the three substrates (27-29),
Hoping to improve on the overall yield of the latter
process, we wondered if replacement of the tert-butyl

Synthesis of BILA 2157 BS
J . Org. Chem., Vol. 64, No. 18, 1999 6627
Sch em e 9
Ta ble 1
In h ibitor Ba ck bon e Assem bly. Two alternative
routes were evaluated for assembly of the inhibitor
backbone and are depicted in Scheme 9. The first one
uses resolved acid (S)-19, which was coupled to aminodiol
412,13 using DCC-HOBt.22 The resulting diol 30 was
obtained in 92% yield as a pure (>99% homogeneous)
solid. Subsequent cleavage of the tert-butyl ester with
TFA was accompanied by the formation of small amounts
of trifluoroacetylated products. Consequently, the crude
acid was submitted to alkaline treatment prior to isola-
tion, and acid 31 was then isolated in near-quantitative
yield as a white crystalline solid. Completion of the
backbone assembly using this route involved coupling
with P4-P3 (2), conveniently carried out using pivaloyl
chloride for activation of the carboxyl group of 31. No side
reactions implicating the free hydroxyl groups were
noticed, and compound 6 was obtained in 87% yield after
crystallization (>98.5% homogeneity by HPLC). The
overall yield of 6 starting from (R,S)-15 was 38%.
The second assembly route shown in Scheme 9 uses
resolved acid (S)-22 obtained as shown in Scheme 6.
Thus, coupling of the crude acid to aminodiol 4,12,13 using
again DCC-HOBt,22 gave 6 in 73% yield, identical in all
respects to material produced by the first route. The
overall yield of 6 (>98.5% homogeneous) starting from
(R,S)-15 was 23%.
substrate
R
ratioa
product
yieldb (%)
27
28
29
O-t-Bu
OH
P4-P3
6.5:1
5.4:1
6.1:1
30
31
6
60
56
56
a
Ratios determined by HPLC analysis of crude reaction mix-
b
ture. Isolated yield of isomerically homogeneous material.
resulting in modest yields of diols (55-60%) and accept-
able diastereoselectivities (5.5-6.5:1 vs 4:1 for 25). The
use of additives (methylsulfonamide, quinoline, pyri-
dine)19,20 or performing the oxidation under asymmetric
conditions (AD-mix-R or -â)13,20 had no beneficial effect
on the outcome of the reaction. In all cases, the desired
products (6, 30, 31) precipitated out of the reaction
mixture and were conveniently isolated in isomerically
pure form after a simple filtration followed by recrystal-
lization (see the Supporting Information for details on
the preparation of these analytical standards). The
structural identity and integrity of the diols was con-
firmed by comparison (NMR, HPLC, mp, rotation) to
material prepared by coupling of the appropriate (S)-
acids (19 or 22) with authentic aminodiol 412,13 as shown
in Scheme 9 (see below).
While the second route saves introduction of the most
valuable fragment (4) to latter stages in the assembly
(21) To circumvent side product formation associated with oxidation
of the vinylic chloride functionality of compound 27, we attempted its
conversion into the aminothiazolyl heterocycle prior to double bond
dihydroxylation. However, under previously used conditions,10 upon
treatment of 27 with 1 equiv of NBS, the more nucleophilic P1 double
bond underwent attack (presumably from the least hindered side) and
a single isomer of the bromo derivative, with the relative stereochem-
istries shown below, was isolated (tentative structure based on MS
and NMR).
Since the slight improvements in diastereoselection
observed in the dihydroxylation of substrates 29-31 were
not sufficient to overcome problems of chemoselectivity
between double bonds, this approach was abandoned in
favor of our initial and more convergent assembly pro-
tocols, using aminodiol 4 as a presynthesized fragment.21
(19) (a) Schro¨der, M. Chem. Rev. 1980, 80, 187.
(20) Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino, G. A.;
Hartung, J .; J eong, K.-S.; Kwong, H.-L.; Morikawa, K.; Wang, Z.-M.;
Xu, D.; Zhang, X.-L. J . Org. Chem. 1992, 57, 2768.
(22) Bodansky, M. In Principles of Peptide Synthesis; Springler-
Verlag: New York, 1984.

6628 J . Org. Chem., Vol. 64, No. 18, 1999
Beaulieu et al.
Sch em e 10
sequence, the first process delivers 6 with greater overall
efficiency. In addition, many intermediates along that
sequence exhibited favorable physical properties, which
allowed purification by crystallization at several steps
along the sequence. In contrast, the use of the second
route required the processing of crude oily materials from
(R,S)-20 until compound 6, which could then be purified
by crystallization. Nevertheless, material obtained from
either routes was shown by NMR and HPLC to be free
from contamination by P2 epimers (see the Supporting
Information for details on the preparation of analytical
standards).
Deter m in a tion of th e Absolu te Con figu r a tion of
Ster eogen ic Cen ter s. We were successful in obtaining
crystals of 31 suitable for X-ray structure determination
(Figure 2).23 This allowed unambiguous assignment of
the absolute configurations of all stereogenic centers in
the molecule (and therefore BILA 2157 BS), including
the enzymatically generated P2 center. By convergence
of several of the described routes to compound 6, it also
confirmed the relative stereochemistry of the two carbinol
centers that are introduced through the dihydroxylation
protocol (Table 1), as well as establishing the (S)-
configuration of the asymmetric carbon atom of 22,
derived from enzymatic hydrolysis of (R,S)-21.
F igu r e 2.
that the final steps of the synthesis be carried out with
maximum efficiency and control of side-product forma-
tion.
Conversion of the vinyl chloride 6 to BILA 2157 BS
requires four chemical transformations as depicted in
Scheme 10.10 First, protection of the 1,2-diol in the form
of an acetonide (32) is required to prevent side reactions
in the following step. The acetonide is not isolated per
say, but volatiles removed under reduced pressure and
the residue taken up in a biphasic tert-butyl methyl ether
(TBME)/water system. Addition of N-bromosuccinimide
at 0 °C then converts the vinyl chloride to bromomethyl
ketone 33, which is treated in situ with thiourea to
produce aminothiazole 34 as a water-soluble salt.9,10 The
cooled aqueous phase containing 34 is separated, washed
with TBME in the cold (to avoid premature cleavage of
the acetonide), and then heated to 60 °C to affect cleavage
of the acetonide and elimination of residual volatile
organic solvents. Subsequent neutralization with NaOH
Con str u ction of th e Am in oth ia zole Heter ocycle.
Previously, purification of BILA 2157 BS to satisfactory
levels could only be achieved using chromatographic
techniques, due to the poor physical properties (amor-
phous solid) of 1, and our lack of success in forming
crystalline addition salts with achiral/chiral, organic or
mineral acids.10 It was therefore of outmost importance
(23) The authors have deposited atomic coordinates for this struc-
ture with the Cambridge Crystallographic Data Centre. The coordi-
nates can be obtained, on request, from the Director, Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ,
U.K.

Synthesis of BILA 2157 BS
J . Org. Chem., Vol. 64, No. 18, 1999 6629
mL). Sodium methoxide (1143 mL of a 25% w/w solution in
MeOH, 5.00 mol, 1 equiv) was added in portions, followed by
a MeOH rinse (550 mL). The reaction mixture was warmed
to 62 °C, and 2,3-dichloropropene 16 (461 mL, 5.00 mol, 1
equiv) in MeOH (550 mL) was added dropwise over a period
of 24-48 h while the internal temperature of the reaction was
maintained above 65 °C. Additional sodium methoxide solution
(57 mL) was added and the mixture heated to 70 °C for 3 h to
complete the reaction. After the mixture was cooled to ambient
temperature, the white slurry was filtered to remove NaCl,
using MeOH for rinses, and the solvent removed under
reduced pressure. Water (2 L) was added to the solid residue,
and the aqueous phase was separated and extracted with
ether. The organic phases were combined, washed with 1 N
HCl (3 × 500 mL), water, and brine, and dried over MgSO4.
The ether was removed under reduced pressure and the
residue fractionally distilled under vacuum to give recovered
dimethylmalonate (1560 mL, bp 80 °C/1.5-2.0 Torr) containing
some monoalkylated material and monoalkylated malonate 17
(620.3 g, 60% yield, Bp 79-86 °C/0.6 Torr) containing <3%
dialkylated material and <5% dimethylmalonate. The distilled
product was used without further purification in the next step,
but a small sample was purified by flash chromatography for
characterization: Rf 0.49 (4:1 hexane/ether); IR (film on NaCl)
ν 1737, 1637 cm-1; 1H NMR (CDCl3) δ 5.25 (broad m, 1H), 5.23
(broad d, J ) 1.5 Hz, 1H), 3.81 (t, J ) 7.5 Hz, 1H), 3.76 (s,
6H), 2.95 (dd, J ) 7.5, 1 Hz, 2H); 13C NMR (CDCl3) δ 168.6,
138.2, 115.2, 52.7, 49.8, 38.3; MS (ES+) m/z 207 (MH+), 229
(M + Na+).
then induces precipitation of BILA 2157 BS free base in
84% yield from 6 and 94.5% homogeneity (HPLC).
Several factors are critical to the success of this
integrated four-step, one-pot sequence that delivers crude
1 with ca. 5% loss in homogeneity with respect to the
starting material 6. The biphasic system used in the NBS
reaction is superior to the originally reported homoge-
neous conditions (aqueous acetonitrile)10 because contact
of the sensitive bromoketone 33 with water is minimized.
In addition, washing of the aqueous hydrobromide solu-
tion of 34 prior to cleavage of the acetonide also serves
to improve the purity of the final product.
Further purification of BILA 2157 BS could be carried
out by conversion to its water-soluble dimesylate salt,
followed by washing with dichloromethane and repre-
cipitation of the free base with aqueous NaOH. After two
such treatments, homogeneity was increased by ca. 3%
to ∼97.5%, with an 80% recovery of the material. BILA
2157 BS obtained by this route was comparable to
material obtained previously10 and was contaminated by
less than 0.01% of its epimer at P2 (HPLC).
In preliminary toxicology studies, chronic administra-
tion of BILA 2157 BS in rats and dogs resulted in
toxicological findings, which precluded further develop-
ment.
Recovered dimethylmalonate (1433 mL) was combined with
fresh material (2000 mL) and alkylated under the conditions
described above. Following three such alkylation cycles, the
combined yield of distilled 17 was 70%.
Con clu sion
BILA 2157 BS (1), a potent and orally active renin
inhibitor, was prepared by a highly convergent 15-step
synthesis from aminodiol 4. Several chemical steps were
combined through implementation of multistep, inte-
grated sequences and one-pot operations amenable to
large scale. Key features of this synthesis include a
solvent-free malonate alkylation under phase-transfer
conditions and the use of an enzyme for the large-scale
preparation of homochiral succinic acid derivatives (with
recycling of the antipode through racemization). In
addition, previously required cryogenic conditions and the
use of a chiral auxiliary have been eliminated, signifi-
cantly reducing the overall cost for the process. Finally,
the end product was purified to ca. 97% homogeneity
without chromatography.
Dia lk yla ted Ma lon a te 18. A 22 L vessel was equipped
with a mechanical stirrer, thermometer, and dropping funnel
and immersed in an ice-water bath. The flask was charged
with 10 N NaOH (13 L), the solution was cooled to 10 °C, and
benzyltriethylammonium chloride (11.58 g, 51 mmol, 0.004
equiv) was added. A mixture of malonate 17 (2627 g, 12.72
mol) and tert-butyl bromoacetate (2480 g, 12.72 mol) was added
dropwise, maintaining the internal reaction temperature below
15 °C. The reaction mixture was then left stirring overnight
at room temperature. The organic layer was separated and
the aqueous phase extracted with hexane. This extract was
evaporated under reduced pressure, the residue combined with
the main organic layer, and the mixture used directly in the
next step. A small sample was purified as follows for charac-
terization: the crude material was diluted with ether and the
solution washed with 1 N NaOH, 1 N HCl, and brine. After
the solution was dried over MgSO4, the solvent was removed
under reduced pressure and the residue purified by flash
chromatography on silica gel using 5% EtOAc in hexane as
eluent. Compound 18 was obtained as a colorless oil: Rf 0.31
(10% EtOAc/hexane); IR (film on NaCl) ν 1741, 1734, 1632
cm-1; 1H NMR (CDCl3) δ 5.32 (broad d, J ) 1.5 Hz, 1H), 5.21
(broad s, 1H), 3.75 (s, 6H), 3.24 (s, 2H), 3.06 (s, 2H), 1.43 (s,
9H); 13C NMR (CDCl3) δ 169.9, 169.5, 137.1, 117.6, 81.3, 54.4,
52.9, 41.8, 37.4, 27.9; MS (ES+) m/z 321 (MH+), 343 (M + Na+).
Anal. Calcd for C14H21ClO6: C, 52.42; H, 6.60. Found: C, 52.19;
H, 6.73.
Ra cem ic Meth ylsu ccin a te (R,S)-15. Crude malonate
derivative 18 from above (assume 12.72 mol) was diluted with
MeOH (7 L) in a 22 L vessel equipped with a thermometer,
mechanical stirrer, and addition funnel. The solution was
cooled to 10 °C in an ice-water bath, and a solution of KOH
(1000 g, 17.80 mol) in water (4 L) was added at such a rate to
maintain the reaction temperature below 15 °C. After comple-
tion, the mixture was stirred overnight at room temperature.
MeOH was then removed under reduced pressure and the
aqueous phase washed with hexane (4 × 1 L). The aqueous
layer was acidified to pH 1 with concentrated HCl (1.4 L) and
saturated with NaCl (500 g). The product was extracted with
toluene (3 × 2 L), and the extracts were washed with brine,
dried over MgSO4, and filtered. The filtrate was then refluxed
for 20 h to affect decarboxylation. The toluene was then
Exp er im en ta l Section
Gen er a l Meth od s. All reagents, solvents, and starting
materials were obtained from commercial sources and used
as received. Benzotriazol-1-yl-1,1,3,3-tetramethyluronium tet-
rafluoroborate (TBTU)24 was purchased from Richelieu Bio-
technologies Inc. (Montre´al, Canada). Alcalase 2.4 L (food
grade) was obtained from Novo Nordisk Biochem.18a 1H NMR
spectra were recorded at 400 MHz and 13C NMR at 100 MHz.
Flash chromatography25 was performed on Merck silica gel 60
(0.040-0.063 mm). Analytical thin-layer chromatography
(TLC) was carried out on precoated (0.25 mm) Merck silica
gel F-254 plates.
All reactions requiring anhydrous conditions were conducted
under a positive pressure of argon or nitrogen gas, in oven-
dried glassware.
Dim eth yl (2-Ch lor o-2-p r op en yl)m a lon a te 17. A 10 L
vessel, equipped with a heating mantle, mechanical stirrer,
thermometer, reflux condenser, and addition funnel, was
purged with dry nitrogen gas and charged with dimethylma-
lonate (2642 g, 20.00 mol, 4 equiv) and anhydrous MeOH (550
(24) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahe-
dron Lett. 1989, 30, 1927.
(25) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923.

6630 J . Org. Chem., Vol. 64, No. 18, 1999
Beaulieu et al.
evaporated under reduced pressure, the residue suspended in
2 N HCl, and the product extracted into hexane. After being
washed with water and brine, the solution was dried (Na2-
SO4), filtered, and evaporated. The residue was distilled under
vacuum to give pure succinate (R,S)-15 as an oil (2664 g, 79%
yield from 17): bp 130-135 °C/0.75 Torr; Rf 0.34 (10% EtOAc/
hexane); IR (film on NaCl) ν 1733, 1635 cm-1; 1H NMR (CDCl3)
δ 5.24 (broad d, J ) 1 Hz, 1H), 5.19 (broad m, 1H), 3.71 (s,
3H), 3.14 (m, 1H), 2.76 (ddd, J ) 14.5, 6.5, 1 Hz, 1H), 2.59
(dd, J ) 16.5, 8.5 Hz, 1H, part of ABX), 2.54 (ddd, J ) 14.5, 8,
0.5 Hz, 1H), 2.48 (dd, J ) 16.5, 5.5 Hz, 1H, part of ABX), 1.44
(s, 9H); 13C NMR (CDCl3) δ 174.1, 170.5, 139.2, 115.0, 81.0,
51.9, 40.7, 39.2, 36.0, 28.0; MS (FAB) m/z 263 (MH+). Anal.
Calcd for C12H19ClO4: C, 54.86; H, 7.29. Found: C, 54.64; H,
7.42. HPLC (ChiralPak AS 4.6 × 250 mm, 0.25% EtOH/
hexane, 0.5 mL/min flow rate): (R)-15 tR 9.6 min (50%); (S)-
15 tR 10.6 min (50%).
and removal of solvent under reduced pressure, the residue
was distilled as previously described to give (R,S)-15 (293.47
g, 97% yield). This material was recycled through enzymatic
hydrolysis.
P r ep a r a tion of Mon om eth ylsu ccin a te (R,S)-20. Race-
mic diester (R,S)-15 (607.76 g, 2.31 mol) was added in portions
to cold (5 °C) 4 N HCl in dioxane (1.6 L) and the mixture
stirred for 40 h, allowing the temperature to raise to ambient.
Volatiles were then removed in a vacuum at 40 °C to give a
yellow oily residue. Ether (250 mL) was added followed by
hexane until slightly turbid and the solution allowed to
crystallize, first at room temperature and then at 5 °C
overnight. The product was collected by filtration, washed with
10% ether/hexane, followed by hexane, and dried under
vacuum to constant weight, to give (R,S)-20 (407.46 g, 85%
yield): mp 61-64 °C; IR (NaCl film) ν 3600-2500, 1736, 1713,
1636 cm-1; 1H NMR (CDCl3) δ 11.45 (broad s, 1H), 5.27 (broad
d, J ) 1 Hz, 1H), 5.22 (broad s, 1H), 3.72 (s, 3H), 3.20 (m,
1H), 2.80 (ddd, J ) 14.5, 6, 1 Hz, 1H, part of ABX), 2.74 (dd,
J ) 17, 8.5 Hz, 1H, part of ABX), 2.62 (dd, J ) 17.5, 5 Hz, 1H,
En zym a tic Hyd r olysis of (R,S)-15. Isola tion of Acid
(S)-19 a n d Recover y of Ester (R)-15. A 12 L vessel equipped
with a mechanical stirrer and a glass pH electrode was charged
with water (4.25 L), acetone (260 mL), and (R,S)-15 (2660 g,
10.12 mol). Alcalase 2.4 L (54 g, Novo Nordisk food grade
enzyme)18a was added, and the pH of the mixture was adjusted
to 7.2-7.5 by slow addition of 2 N NaOH (∼65 mL). The
reaction mixture was then stirred vigorously, and the pH of
the mixture maintained at 7.2-7.5 by addition of 2 N NaOH
using an automatic titration device (pH meter coupled to a
peristaltic addition pump). After 26 h, the pH of the mixture
was raised and maintained at 7.5-7.8 for an additional 22 h,
using 2 N NaOH. The pH was then readjusted once more to
7.8-8.1 and maintained by addition of 2 N NaOH for another
23 h. The total amount of added 2 N NaOH, after this point,
was 2.38 L (4.76 mol, 94% of theoretical amount).
part of ABX), 2.58 (dd, J ) 14.5, 8.5 Hz, 1H, part of ABX); 13
C
NMR (CDCl3) δ 177.3, 173.7, 138.6, 115.2, 52.0, 40.3, 38.5, 33.9;
MS (FAB) m/z 207 (MH+). Anal. Calcd for C8H11ClO4: C, 46.50;
H, 5.37. Found: C, 46.12; H, 5.35.
Cou p lin g of (R,S)-20 w it h Am in e 2. P r ep a r a t ion of
(R,S)-21. Acid (R,S)-20 (138.00 g, 667.9 mmol) and N-meth-
ylmorpholine (147 mL, 1.34 mol) were dissolved in dry THF
(500 mL), and the solution was cooled in an ice-water bath
under an argon atmosphere. Pivaloyl chloride (81.5 mL, 663
mmol) was added dropwise over a 30 min period and stirring
continued for an additional 1 h at 5 °C. Amine 210 (191.0 g,
0.660 mmol) in THF (75 mL + 25 mL rinse) was added over
30 min and the mixture stirred for 1.5 h at 5 °C. The reaction
mixture was then quenched with water (100 mL) and THF
evaporated under reduced pressure. The product was extracted
with EtOAc, washed with water and brine, dried (MgSO4/
charcoal), and concentrated to give crude (R,S)-21 (323 g, 102%
yield) as a thick orange oil, which gave the following analytical
data: Rf 0.24 (9:1 EtOAc/MeOH); IR (film on NaCl) ν 1730,
1660, 1640 cm-1; 1H NMR (CDCl3, mixture of rotamers) δ 8.62
(broad m, 0.1 H), 8.58 (m, 0.4H), 8.54 (broad d, J ) 5 Hz, 0.6H),
7.62 (m, 1H), 7.26-7.13 (m, 2H), 5.26-5.15 (m, 2H), 4.20 (d,
J ) 16 Hz, 0.5H), 4.10 (d, J ) 16 Hz, 0.4H), 4.15-4.06 (m,
0.1H), 4.01-3.89 (m, 0.1H), 3.96 (d, J ) 16 Hz, 0.4H), 3.84 (d,
J ) 16 Hz, 0.5H), 3.80-3.65 (m, 2H), 3.70 (s, 1.5H), 3.69 (s,
1.5H), 3.27 (m, 1H), 3.17 (m, 1H), 3.11-2.94 (m, 3H), 2.92 (s,
1.8H), 2.90 (s, 1.2H), 2.82-2.71 (m, 1.9H), 2.71-2.59 (m, 1.9H),
2.38 (m, 0.1H), 2.24 (m, 0.1H), 1.80-1.35 (m, 6H), 1.20 (m,
The reaction mixture was extracted with ether, and the
combined organic layers were extracted with pH 9 aqueous
NaOH (1 L, save extract), washed with brine, dried (Na2SO4),
and concentrated to provide (R)-15 as a pure colorless oil (1349
1
g, 51% yield): Rf, IR, H NMR, 13C NMR, MS (FAB) identical
to that of (R,S)-15: [R]25D +6.7° (c 1.05, MeOH); [R]25365 +14.4°
(c 1.05, MeOH). Anal. Calcd for C12H19ClO4: C, 54.86; H, 7.29.
Found: C, 54.92; H, 7.33. HPLC (ChiralPak AS 4.6 × 250 mm,
0.25% EtOH/hexane, 0.5 mL/min flow rate): (R)-15 tR 9.8 min
(97.2%, 94.4% ee); (S)-15 tR 11.0 min (2.8%).
The aqueous phase from the enzymatic hydrolysis was
combined with the pH 9 NaOH wash from the isolation of the
(R)-ester (above) and acidified to pH 1 with concentrated HCl
(500 mL). The mixture was then extracted with ether and the
extract washed with brine and dried (Na2SO4). Removal of
volatiles under reduced pressure gave pure acid (S)-19 as a
clear yellowish oil (1213 g, 48% yield): Rf 0.69 (60% EtOAc/
13
2H), 0.90 (m, 2H); C NMR (CDCl3, mixture of rotamers) δ
182.4, 174.8, 174.7, 171.3, 171.2, 167.8, 167.7, 167.4, 167.2,
158.8, 158.6, 158.1, 158.0, 149.6, 149.3, 148.9, 148.8, 139.6,
139.5, 137.0, 136.9, 136.88, 136.85, 124.0, 123.9, 123.8, 123.7,
122.2, 121.9, 121.7, 121.6, 114.9, 114.7, 114.6, 77.2, 55.1, 54.9,
53.7, 53.6, 51.9, 51.8, 51.7, 50.0, 49.6, 48.75, 48.7, 48.1, 47.4,
47.1, 40.7, 40.65, 40.6, 39.6, 39.4, 38.4, 37.1, 37.0, 36.5, 36.4,
36.2, 35.6, 35.5, 35.0, 34.9, 33.5, 33.4, 33.35, 33.3, 33.2, 33.1,
31.0, 30.93, 30.90, 30.85, 30.8, 30.75, 30.7, 30.6, 27.1, 26.4,
26.35, 26.3, 25.8; MS (FAB) m/z 478 (MH+). Anal. Calcd for
hexane); [R]25 -4.7° (c 1.02, MeOH); [R]25
-6.7° (c 1.02,
D
365
MeOH); IR (film on NaCl) ν 3336-2617, 1732, 1716, 1636 cm-1
;
1H NMR (CDCl3) δ 5.26 (d, J ) 1 Hz, 1H), 5.22 (broad s, 1H),
3.17 (m, 1H), 2.81 (broad dd, J ) 14.5, 6 Hz, 1H), 2.63-2.49
(m, 3H), 1.44 (s, 9H); 13C NMR (CDCl3) δ179.7, 170.4, 138.9,
115.3, 81.3, 40.3, 39.1, 35.5, 28.0; MS (FAB) m/z 249 (MH+).
Anal. Calcd for C11H17ClO4: C, 53.12; H, 6.89. Found: C, 52.86;
H, 7.01. HPLC of methyl ester prepared with diazomethane
in ether (ChiralPak AS 4.6 × 250 mm, 0.25% EtOH/hexane,
0.5 mL/min flow rate): (R)-15 tR 9.9 min (1%); (S)-15 tR 10.9
min (99%, 98% ee).
C
25H36ClN3O4: C, 62.82; H, 7.59; N, 8.79. Found: C, 62.46; H,
7.71; N, 8.50.
En zym a tic Hyd r olysis of (R,S)-21. Isola tion of Acid
(S)-22 a n d Recover y of Ester (R)-21. Racemic succinate
(R,S)-21 (196.2 g, 0.41 mol) was dissolved in acetone (150 mL)
and the solution diluted with water (350 mL). The pH of the
solution was adjusted to 7 by addition of 1 M KHSO4, and 5 g
of Alcalase 2.4 L18a was added. The solution was stirred
vigorously and its pH maintained between 7.5 and 7.8 for 15
h by addition of 1 N NaOH (∼130 mL added over this time
period, ∼75% of theoretical amount) using an automatic
titration device (pH meter coupled to a peristaltic addition
pump). The pH of the solution was then raised to 8 and
maintained for another 24 h (∼30 mL of 1 N NaOH, 92% of
theoretical amount). After a total of 160 mL of 1 N NaOH had
been added, the reaction mixture was concentrated under
reduced pressure to remove acetone and the aqueous phase
Ra cem iza tion of (R)-15. Methyl ester (R)-15 (300.70 g,
1.144 mol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 8.7
g, 57 mmol, 0.05 equiv) were stirred at 80 °C under a nitrogen
atmosphere for 24 h. HPLC analysis of an aliquot (partitioned
between EtOAc/1 N HCl) on a chiral support showed almost
complete racemization (ChiralPak AS 4.6 × 250 mm, 0.25%
EtOH/hexane, 0.5 mL/min flow rate): (R)-15 tR 10.6 min (53%);
(S)-15 tR 12.1 min (47%). The reaction mixture was then cooled
to room temperature and diluted with EtOAc (500 mL) and
the solution washed with 1 N HCl (3 × 300 mL). The aqueous
phase was extracted with EtOAc, and the organic extracts
were combined and washed with brine. After drying (Na2SO4)

Synthesis of BILA 2157 BS
J . Org. Chem., Vol. 64, No. 18, 1999 6631
extracted with EtOAc. The aqueous phase was saved for
recovery of (S)-22 (see below). The organic extract was washed
with water (2 × 100 mL) and brine and dried (MgSO4/
charcoal). The solution was then filtered through a small pad
of silica gel using EtOAc (1 L) for washings. Removal of solvent
under reduced pressure followed by drying under vacuum to
constant weight gave crude (R)-21 as a thick brown oil (103.69
g, 53% yield): [R]25 -2.5° (c 1.27, MeOH); [R]25 -15.0° (c
Ra cem iza tion of (R)-21. NaOMe (25% w/w solution in
MeOH, 152 µL, 0.66 mmol, 0.1 equiv) was added to a solution
of (R)-21 (3.19 g, 6.67 mmol) in MeOH (10 mL). The dark red
solution was stirred under reflux (60 °C) for 18 h, at which
point HPLC analysis on a chiral support as described above
for the pure enantiomers indicated complete racemization.
Cou p lin g of Acid (S)-22 w ith Am in e 4. P r ep a r a tion of
Com p ou n d 6. Aminodiol 412,13 (2.00 g, 8.2 mmol), acid (S)-22
(4.22 g, 9.1 mmol), hydroxybenzotriazole (1.50 g, 9.8 mmol),
and N-methylmorpholine (5.00 g, 49.5 mmol) were dissolved
in dry DMF (20 mL). Dicyclohexylcarbodiimide (1.90 g, 9.2
mmol) was added and the mixture stirred overnight at room
temperature. The reaction mixture was then concentrated to
half volume under vacuum and subsequently diluted with
EtOAc (500 mL). Precipitated DCU was removed by filtration
and the filtrate washed with water and brine. After drying
(MgSO4) and removal of solvent under reduced pressure a
yellow solid was obtained. The crude material was dissolved
in boiling EtOAc (20 mL), hexane (100 mL) was added, and
the product was allowed to crystallize at room temperature.
After cooling (5 °C), the material was collected by filtration
and washed with 10% EtOAc/hexane and then with hexane.
After drying in a vacuum, compound 6 was obtained as a white
D
365
1.27, MeOH). HPLC analysis (two end to end Chiralcel OD-H
columns of 15 and 25 cm, 2% EtOH/hexane isocratic, 0.5 mL/
min flow rate, 260 nm UV detection): (R)-21 tR 154 min (98.8%,
97.6% ee); (S)-21 tR 171 min (1.2%). An analytical sample of
this material was obtained by flash chromatography on silica
gel using 2% MeOH/CHCl3 then 3% MeOH/CHCl3. (R)-21, was
obtained as a colorless oil: Rf 0.34 (5% MeOH/CHCl3); [R]25
D
-2.9° (c 1.24, MeOH); [R]25365 -18.1° (c 1.24, MeOH); IR (NaCl
film) ν 2924, 2851, 1736, 1644 cm-1; 1H NMR (CDCl3, mixture
of rotamers) δ 8.59 (broad d, J ) 5 Hz, 0.1H), 8.55 (dt, J ) 4,
1.5 Hz, 0.4H), 8.50 (broad d, J ) 3 Hz, 0.5H), 7.66-7.56 (m,
1H), 7.23-7.09 (m, 2H), 5.26-5.13 (m, 2H), 4.19 (d, J ) 16
Hz, 0.5H, part of ABX), 4.10 (d, J ) 16 Hz, 0.4H, part of ABX),
4.08 (d, J ) 17 Hz, 0.1H, part of ABX), 3.99 (d, J ) 17 Hz,
0.1H, part of ABX), 3.95 (d, J ) 16 Hz, 0.5H, part of ABX),
3.83 (d, J ) 16 Hz, 0.4H, part of ABX), 3.79-3.66 (m, 2H),
3.69 (s, 1.5H), 3.67 (s, 1.5H), 3.31-2.95 (m, 5H), 2.90 (broad
s, 3H), 2.86-2.50 (m, 3.5H), 2.39 (d, J ) 6 Hz, 0.1H), 2.35 (d,
J ) 6.5 Hz, 0.1H), 2.24 (m, 0.5H), 1.80-1.36 (m, 6H), 1.30-
1.08 (m, 3H), 0.98-0.80 (m, 2H); 13C NMR (CDCl3, mixture of
rotamers) δ 174.8, 174.7, 171.3, 171.1, 167.7, 167.6, 167.3,
167.1, 159.1, 158.8, 158.2, 157.7, 148.7, 149.5, 149.2, 149.1,
139.61, 139.58, 139.55, 139.5, 136.8, 136.6, 136.5, 136.4, 123.8,
123.7, 123.6, 123.5, 122.1, 121.7, 121.5, 121.4, 114.8, 114.6,
114.5, 55.0, 54.8, 53.7, 53.6, 51.82, 51.80, 51.7, 50.0, 49.61, 48.7,
48.6, 48.5, 47.9, 47.3, 47.0, 40.7, 40.65, 40.60, 39.5, 39.3, 37.1,
37.0, 36.6, 36.4, 36.3, 36.2, 35.9, 35.8, 35.0, 34.9, 33.5, 33.3,
33.25, 33.2, 33.1, 31.0, 30.9, 30.85, 30.8, 30.75, 30.70, 30.6,
26.37, 26.35, 26.27, 26.26, 25.8; MS (ES+) m/z 478 (MH+). Anal.
Calcd for C25H36ClN3O4: C, 62.82; H, 7.59; N, 8.79. Found: C,
62.51; H, 7.89; N, 8.75.
1
solid (4.16 g, 73% yield): Rf, IR, H NMR, 13C NMR, and MS
data are identical to that of 6 obtained by coupling of acid 31
with amine 4: mp 139-141 °C; [R]24 -32.2° (c 1.00, MeOH).
D
Anal. Calcd for C38H61ClN4O5: C, 66.21; H, 8.92; N, 8.13.
Found: C, 65.99; H, 9.02; N, 8.09. Reversed-phase HPLC
(Supelcosil LC-ABZ, 4.6 × 150 mm, 5 µm particle size, 0-100%
0.1% TFA-CH3CN in 0.1% TFA in 25 min then 100% 0.1%
TFA in CH3CN for 5 min, 1 mL/min flow rate, UV detection
at 215 nm): tR 17.0 min (99.04%, P2 epimer of 6 not detected).
P r ep a r a tion of Allylic Am in e Hyd r och lor id e 26. A 4:1
v/v mixture of AcOH/12 N HCl (200 mL) was slowly added
(caution: gas evolution) to carbamate 2513 (154.22 g, 0.498
mol), and the mixture was stirred for 1.5 h at room temper-
ature. An additional 300 mL of the acid mixture was added
and the solution stirred for another 2 h, after which point TLC
(20% EtOAc/hexane) showed complete disappearance of start-
ing material. The reaction mixture was brought to pH 14 by
slow addition of 10 N NaOH (850 mL) and extracted with
ether. The combined extracts were washed with 1:1 water/
brine, dried (Na2SO4), and concentrated to a viscous black oil.
This residue was dissolved in ether (100 mL), and a 1 N
solution of hydrogen chloride in ether (500 mL) was added
dropwise over 1 h. The resulting slurry was concentrated by
removal of ether (300 mL) under reduced pressure, hexane
(200 mL) was added in portions, and the mixture was stirred
for 30 min. The precipitated hydrochloride salt was collected
by filtration and washed with cold 25% hexane in ether (2 ×
250 mL) and then 50% hexane in ether (2 × 250 mL). After
drying in air, hydrochloride 26 was obtained as a tan-colored
The aqueous layer from the first EtOAc extraction (see
above) was acidified to pH 4 by addition of 20% aqueous
KHSO4 and extracted with EtOAc. The extract was filtered to
remove precipitated enzyme residues and washed with water
and brine. After drying (MgSO4), concentration, and drying
to constant weight under vacuum, crude acid (S)-22 (67.35 g,
75% yield) was obtained as a brown foam: [R]25D +5.1° (c 1.30,
MeOH); [R]25365 +23.8° (c 1.30, MeOH). A small sample of this
crude acid was converted to the corresponding methyl ester
(S)-21 using diazomethane and analyzed by HPLC on a chiral
support (two end to end Chiralcel OD-H columns of 15 and 25
cm, 2% EtOH/hexane isocratic, 0.5 mL/min flow rate, 260 nm
UV detection): (R)-21 tR 157 min (3%); (S)-21 tR 167 min (97%,
94% ee). An analytical sample of acid (S)-22 was obtained by
flash chromatography on silica gel using 5% MeOH/CHCl3 as
eluent: Rf 0.19 (5% MeOH/CHCl3); [R]25D +5.2° (c 1.33, MeOH);
solid (104.59 g, 85% yield): mp 220-227 °C dec; [R]25 +22.6°
D
(c 1.04, MeOH); IR (NaCl film) ν 1601 cm-1; 1H NMR (CDCl3,
contains ∼5% of E-isomer) δ 8.37 (broad s, 3H), 5.83 (m, 0.05H,
E-isomer), 5.72 (m, 0.95H), 5.45 (m, 1H), 4.09 (broad s, 0.95H),
3.75 (broad s, 0.05H, E-isomer), 2.13-2.03 (m, 1H), 2.02-1.92
(m, 1H), 1.82-1.52 (m, 8H), 1.42-1.06 (m, 4H), 1.00-0.86 (m,
2H), 0.94 (d, J ) 6.5 Hz, 3H), 0.90 (d, J ) 6.5 Hz, 3H); 13C
NMR (CDCl3) δ 135.9, 126.0, 47.0, 41.2, 36.8, 33.8, 33.5, 32.7,
28.4, 26.3, 26.0, 25.9, 22.5, 22.2; MS (ES+) m/z 210 (MH+).
Anal. Calcd for C14H28ClN: C, 68.40; H, 11.48; N, 5.70.
Found: C, 68.23; H, 11.70; N, 5.73.
[R]25 +25.9° (c 1.33, MeOH); IR (NaCl film) ν 3700-2300,
365
1726, 1657, 1642 cm-1; 1H NMR (CDCl3, mixture of rotamers)
δ 10.25 (broad s, 1H), 8.61 (broad d, J ) 5 Hz, 0.2H), 8.59
(broad d, J ) 4.5 Hz, 0.3H), 8.55 (broad d, J ) 5 Hz, 0.5 H),
7.66 (dt, J ) 7.5, 2 Hz, 1H), 7.28-7.15 (m, 2H), 5.31-5.18 (m,
2H), 4.12-3.84 (m, 2H), 3.78-3.62 (m, 2H), 3.28-3.00 (m,
4.5H), 2.97 (d, J ) 5 Hz, 0.1H), 2.93 (s, 1.6H), 2.90 (s, 1.4H),
2.90-2.77 (m, 0.5H), 2.76-2.70 (m, 1.3H), 2.68-2.43 (m, 0.8H),
2.43-2.25 (m, 0.2H), 1.80-1.37 (m, 6H), 1.30-1.10 (m, 3H),
0.98-0.80 (m, 2H); 13C NMR (CDCl3, mixture of rotamers) δ
176.1, 176.0, 175.95, 172.7, 172.4, 172.2, 167.62, 167.61, 167.1,
167.0, 158.5, 158.3, 157.8, 157.4, 149.3, 149.0, 148.2, 148.1,
139.9, 139.8, 137.5, 137.4, 137.3, 124.15, 124.10, 124.0, 122.3,
122.0, 121.9, 121.7, 115.0, 114.9, 114.6, 114.5, 55.4, 55.1, 54.0,
53.9, 50.2, 49.9, 48.7, 48.6, 48.1, 47.6, 47.5, 40.5, 40.4, 40.35,
40.3, 39.6, 39.4, 39.3, 37.0, 36.9, 36.3, 36.15, 36.10, 35.9, 35.2,
35.1, 34.8, 33.5, 33.3, 33.15, 33.10, 33.0, 32.8, 30.9, 30.7, 30.6,
26.35, 26.30, 26.2, 25.7; MS (ES+) m/z 464 (MH+). Anal. Calcd
for C24H34ClN3O4: C, 62.13; H, 7.39; N, 9.06. Found: C, 62.12;
H, 7.65; N, 8.98.
Cou p lin g of Acid (S)-19 w ith Am in e Hyd r och lor id e 26.
P r ep a r a tion of Alk en e 27. To a slurry of hydrochloride salt
26 (10.00 g, 40.7 mmol) in acetonitrile (50 mL) were added
diisopropylethylamine (28.3 mL, 163 mmol) and a solution of
acid (S)-19 (10.12 g, 40.7 mmol) in acetonitrile (25 mL). TBTU
(14.36 g, 44.8 mmol) was added and the mixture stirred 18 h
at room temperature. Volatiles were then removed under
reduced pressure and the residue dissolved in ether (250 mL).
The organic solution was washed with 2.5 N NaOH (2 × 100
mL), 1 N HCl (100 mL), and brine. After drying (MgSO4) and
evaporation of solvents, the residue was purified by flash
chromatography on silica gel using 5% EtOAc in hexane as

6632 J . Org. Chem., Vol. 64, No. 18, 1999
Beaulieu et al.
eluent to provide alkene 27 as a light yellow, waxy solid (15.48
g, 86% yield): mp 60.0-61.5 °C; Rf 0.47 (15% EtOAc/hexane);
130.9, 130.8, 130.7, 130.6, 123.8, 123.7, 123.6, 123.5, 122.1,
121.8, 121.6, 121.5, 114.8, 114.7, 114.5, 114.3, 55.1, 54.9, 53.9,
53.8, 50.0, 49.7, 48.7, 48.5, 47.9, 47.5, 47.1, 44.6, 44.5, 44.4,
44.3, 43.4, 43.3, 41.5, 41.4, 40.6, 40.5, 40.4, 40.3, 37.1, 37.0,
36.7, 36.6, 36.5, 36.3, 36.0, 35.8, 35.3, 35.2, 35.1, 35.0, 34.9,
34.0, 33.9, 33.5, 33.4, 33.3, 33.2, 33.2, 33.1, 33.0, 31.0, 30.9,
30.8, 30.7, 30.6, 30.5, 28.5, 26.5, 26.3, 26.2, 26.1, 25.8, 25.7,
22.3, 22.2; MS (ES+) m/z 655 (MH+), 677 (M + Na+). Anal.
Calcd for C38H59ClN4O3: C, 69.64; H, 9.07; N, 8.55. Found: C,
69.46; H, 9.47; N, 8.48.
Gen er a l P r oced u r e for Dih yd r oxyla t ion of Alk en es
27-29. To the alkene substrate (∼10 mmol, 1 equiv) in tert-
butylmethyl ether (20 mL) were added water (3 equiv) and
N-methylmorpholine N-oxide (1.5 equiv). The solution was
cooled to 5 °C, and OsO4 (0.01 equiv of a 2.5% w/w solution in
tert-butyl alcohol, 1.25 mL/10 mmol of substrate) was added.
The mixture was stirred 48 h at 5 °C, after which volatiles
were removed under reduced pressure. Warm acetonitrile (20
mL for a 10 mmol scale) was added to dissolve solids, followed
by dropwise addition of water (10 mL) to precipitate diol
product. After the mixture was stirred for 0.5 h at room
temperature, precipitated solids were collected by filtration,
washed with 2:1 CH3CN/water (20 mL), and dried. Crude
materials obtained in this way were comparable with products
prepared from authentic aminodiol 4.12,13
[R]25 +21.8° (c 1.02, MeOH); IR (film on NaCl) ν 3285, 1726,
D
1639 cm-1
;
1H NMR (CDCl3) δ 5.66 (d, J ) 8 Hz, 1H), 5.43
(ddt, J ) 11, 7.5, 0.5 Hz, 1H), 5.22-5.16 (m, 3H), 4.73 (m, J )
8 Hz, 1H), 2.91-2.82 (m, 1H), 2.65 (dd, J ) 14, 8 Hz, 1H, part
of ABX), 2.62 (dd, J ) 17, 10 Hz, 1H, part of ABX), 2.38 (dd,
J ) 14, 7 Hz, 1H, part of ABX), 2.33 (dd, J ) 17, 4 Hz, 1H,
part of ABX), 2.02 (m, 2H), 1.78-1.55 (m, 7H), 1.44 (s, 9H),
1.31-1.10 (m, 5H), 0.97-0.87 (m, 2H), 0.90 (d, J ) 6.5 Hz,
3H), 0.88 (d, J ) 6.5 Hz, 3H); 13C NMR δ 171.6, 171.4, 139.3,
131.3, 130.9, 115.1, 80.9, 44.5, 43.6, 41.6, 40.4, 37.0, 36.7, 34.0,
33.4, 33.3, 28.5, 28.1, 26.5, 26.2, 26.1, 22.4, 22.3. MS (ES+)
m/z 440 (MH+), 462 (M + Na+), 485 (M + 2Na+). Anal. Calcd
for C25H42ClNO3: C, 68.23; H, 9.62; N, 3.18. Found: C, 68.43;
H, 9.88; N, 3.36.
P r ep a r a tion of Acid 28. A 30% solution of HBr in AcOH
(50 mL) was cooled to 5 °C, and ester 27 (8.00 g, 8.2 mmol)
was added. The mixture was stirred overnight at 5 °C.
Volatiles were then removed under vacuum, and the residue
was coevaporated with toluene (2 × 100 mL) under reduced
pressure. The resulting oil was dissolved in ether (300 mL)
and washed with water. The aqueous phase was basified to
pH 7 with 1 N NaOH, and this solution was used again to
wash the original ether layer. This extraction/neutralization
operation was repeated until the aqueous phase remained at
pH 6-7. The ether layer was then dried (MgSO4) and concen-
trated and the residue purified by flash chromatography on
silica gel using 5% MeOH/CHCl3 as eluent. Acid 28 was
obtained as a beige syrup that was dried at 75 °C under
vacuum and solidified on standing (6.16 g, 91% yield): mp
87.2-89.4 °C; Rf 0.31 (5% MeOH/CHCl3); [R]25D + 26.7° (c 1.06,
Diol 30 was obtained as an isomerically pure (NMR) white
solid in 60% yield on a 9 mmol scale. IR, NMR, and MS data
were identical to material prepared by coupling of acid (S)-19
to authentic aminodiol 412,13 (see below): mp 147-148 °C; Rf
0.57 (3:1 hexane/EtOAc); [R]25 -43.4° (c 1.01, MeOH). Anal.
D
Calcd for C25H44ClNO5: C, 63.34; H, 9.35; N, 2.95. Found: C,
63.42; H, 9.74; N, 3.03.
MeOH); IR (film on NaCl) ν 3288, 3072-2720, 1712, 1635 cm-1
;
Diol 31 was obtained as a white solid in 56% yield on a 9.4
mmol scale (contaminated with 3.2% of the epimer at P2 as
determined by 1H NMR comparison with an authentic stan-
dard in DMSO-d6). IR, NMR, and MS data were identical to
material prepared by deprotection of ester 30 (see below): mp
1H NMR (CDCl3) δ 5.65 (d, J ) 8.5 Hz, 1H), 5.45 (dt, J ) 10.5,
7.5 Hz, 1H), 5.21 (broad s, 2H), 5.21-5.14 (m, 1H), 4.76 (m, J
) 8 Hz, 1H), 2.88 (m, 1H), 2.79 (dd, J ) 17, 9 Hz, 1H, part of
ABX), 2.65 (dd, J ) 14, 8 Hz, 1H, part of ABX), 2.52 (dd, J )
17, 3.5 Hz, 1H, part of ABX), 2.46 (dd, J ) 14, 7 Hz, 1H, part
of ABX), 2.01 (tt, J ) 7, 2 Hz, 2H), 1.78-1.56 (m, 6H), 1.44
(broad m, 1H), 1.30-1.10 (m, 5H), 0.98-0.82 (m, 2H), 0.90 (d,
J ) 6.5 Hz, 3H), 0.89 (d, J ) 6.5 Hz, 3H); 13C NMR δ 175.8,
172.1, 138.5, 131.6, 130.4, 115.9, 44.9, 43.4, 41.6, 40.1, 36.7,
35.7, 34.1, 33.4, 33.3, 28.5, 26.5, 26.1, 22.4, 22.3; MS (ES-) m/z
382 (M - H). Anal. Calcd for C21H34ClNO3: C, 65.69; H, 8.93;
N, 3.65. Found: C, 65.48; H, 8.97; N, 3.77.
188-189 °C;. [R]25 -48.5° (c 1.02, MeOH). Anal. Calcd for
D
C
21H36ClNO5: C, 60.35; H, 8.68; N, 3.35. Found: C, 60.57; H,
8.87; N, 3.52.
Diol 6 was obtained as an isomerically pure (NMR) solid
in 56% yield on a 4 mmol scale. IR, NMR, and MS data were
identical to material prepared by coupling of acid 31 with
amine 2 or coupling of acid (S)-22 with aminodiol 412,13 (see
below): mp 138.5-140 °C; Rf 0.47 (5% MeOH/EtOAc); [R]25
D
Cou p lin g of Acid 28 to Am in e 2. P r ep a r a tion of 29. To
a solution of acid 28 (2.250 g, 5.86 mmol) in dry THF (25 mL)
were added N-methylmorpholine (1.93 mL, 17.6 mmol), a
solution of amine 210 (1.866 g, 6.45 mmol) in dry THF (10 mL),
and TBTU (2.069 g, 6.45 mmol). The reaction mixture was then
stirred overnight at room temperature. The mixture was
diluted with ether (200 mL), washed with 2.5 N NaOH (2 ×
25 mL), and concentrated under reduced pressure and the
residue purified by flash chromatography on silica gel using
3% MeOH/CHCl3 as eluent. Compound 29 was obtained as a
yellow glass (3.021 g, 78% yield): Rf 0.23 (5% MeOH/CHCl3);
[R]25 +12.6° (c 1.0, MeOH); [R]25 +48.3° (c 1.0, MeOH); IR
-30.6° (c 1.05, MeOH). Anal. Calcd for C38H61ClN4O5: C, 66.21;
H, 8.92; N, 8.13. Found: C, 65.96; H, 9.21; N, 8.12.
Cou p lin g of (S)-19 to Am in od iol 4. P r ep a r a tion of
Com p ou n d 30. A 5 L vessel fitted with a mechanical stirrer
and thermometer was charged with acid (S)-19 (200.0 g, 804
mmol) and dry THF (500 mL). The solution was cooled to 5 °C
in an ice-water bath, and diisopropylethylamine (154 mL, 885
mmol) was added slowly. Aminodiol 412,13 (195.7 g, 804 mmol),
1-hydroxybenzotriazole (114.1 g, 844 mmol), and additional
THF (500 mL) were then added. A solution of 1,3-dicyclohexyl-
carbodiimide (170.1 g, 824 mmol) in dry THF (300 mL) was
added dropwise to the cooled reaction mixture over a 1 h
period. The white slurry was then stirred overnight at room
temperature. The reaction mixture was diluted with EtOAc
(1 L) and the precipitated dicyclohexylurea (DCU) separated
by filtration (used EtOAc for rinses). The filtrate was then
concentrated under reduced pressure to a white paste that was
transferred to a 5 L three-necked flask and suspended in
acetonitrile (1.9 L). To the vigorously stirred (mechanical
stirrer) suspension was added 2 N HCl (1.6 L) and stirring
continued for 1 h. The precipitated product was collected by
filtration, washed with 1:1 CH3CN/2 N HCl (2 × 500 mL) and
water (3 × 500 mL), and dried in air to constant weight. Diol
30 was obtained as a white solid (353.4 g, 92% yield),
containing 0.6% w/w of DCU and none of the P2 epimer (1H
NMR). An analytical sample of this material was obtained by
filtration through a pad of silica gel using 25% EtOAc/hexane
as eluent: mp 148.5-150 °C; Rf 0.52 (25% EtOAc/hexane);
[R]25D -44.6° (c 1.09, MeOH); IR (KBr) ν 3445, 3251, 1700, 1632
cm-1; 1H NMR (DMSO-d6) δ 7.84 (d, J ) 9 Hz, 1H), 5.36 (broad
D
365
(NaCl film) ν 3315, 1652, 1637 cm-1; 1H NMR (CDCl3, mixture
of rotamers) δ 8.61 (broad d, J ) 5 Hz, 0.1H), 8.57 (broad d, J
) 5 Hz, 0.4H), 8.52 (broad d, J ) 5 Hz, 0.5H), 7.67-7.58 (m,
1H), 7.24-7.11 (m, 2H), 6.22-6.09 (broad m, 1H), 5.47-5.35
(m, 1H), 5.24-5.10 (m, 3H), 4.75-4.63 (m, 1H), 4.35 (dd, J )
16, 13.5 Hz, 0.8H), 4.15 (d, J ) 18 Hz, 0.1H), 4.07 (d, J ) 17.5
Hz, 0.1H), 3.88-3.51 (m, 3H), 3.42 (dd, J ) 13.5, 7.5 Hz, 0.1H,
part of ABX), 3.32 (dd, J ) 13.5, 7.5 Hz, 0.1H, part of ABX),
3.22 (dd, J ) 15, 7.5 Hz, 0.4H, part of ABX), 3.14 (dd, J ) 15,
8 Hz, 0.4H, part of ABX), 3.13-2.98 (m, 3H), 2.97-2.85 (m,
3.5H), 2.81-2-61 (m, 2H), 2.58-2.25 (m, 2.3H), 2.23 (dd, J )
16, 4 Hz, 0.1H, part of ABX), 2.11 (dd, J ) 16, 4 Hz, 0.1H,
part of ABX), 2.07-1.96 (m, 2H), 1.80-1.48 (m, 11H), 1.48-
1.32 (m, 2H), 1.32-1.02 (m, 8H), 1.00-0.75 (m, 4H), 0.89
(broad d, J ) 6.5 Hz, 3H), 0.88 (broad d, J ) 6.5 Hz, 3H); 13
C
NMR δ 172.2, 172.1, 172.0, 171.9, 171.8, 171.7, 167.8, 167.6,
167.3, 167.2, 159.0, 158.7, 158.1, 157.6, 149.8, 149.6, 149.2,
139.9, 139.8, 139.7, 136.8, 136.6, 136.5, 131.2, 131.1, 131.0,

Synthesis of BILA 2157 BS
J . Org. Chem., Vol. 64, No. 18, 1999 6633
d, J ) 1 Hz, 1H), 5.22 (d, J ) 1.5 Hz, 1H), 4.71 (d, J ) 6.5 Hz,
1H), 4.49 (broad d, J ) 4 Hz, 1H), 4.08 (broad dt, J ) 9, 5 Hz,
1H), 3.09-2.99 (m, 2H), 2.92 (broad t, J ) 7.5 Hz, 1H), 2.62
(dd, J ) 14.5, 7 Hz, 1H, part of ABX), 2.44 (dd, J ) 16, 9 Hz,
1H, part of ABX), 2.39 (dd, J ) 14.5, 7 Hz, 1H, part of ABX),
2.23 (dd, J ) 16, 5.5 Hz, 1H, part of ABX), 1.81-1.52 (m, 7H),
1.44 (broad m, 1H), 1.39 (s, 9H), 1.33 (broad m, 1H), 1.25-
1.03 (m, 5H), 0.95-0.71 (broad m, 2H), 0.87 (d, J ) 6.5 Hz,
3H), 0.79 (d, J ) 6.5 Hz, 3H); 13C NMR (CDCl3) δ 174.8, 171.2,
139.3, 115.5, 81.3, 77.6, 69.6, 47.3, 42.6, 41.5, 40.6, 39.5, 37.1,
33.9, 33.7, 32.7, 28.1, 26.5, 26.2, 26.0, 24.6, 23.9, 21.8; MS (ES+)
m/z 474 (MH+), 496 (M + Na+). Anal. Calcd for C25H44ClNO5:
C, 63.34; H, 9.35; N, 2.95. Found: C, 63.44; H, 9.50; N, 3.05.
Dep r otection of Ester 30. P r ep a r a tion of Acid 31. Ester
30 from above (346.2 g, 726 mmol based on DCU content) was
added to ice-cold trifluoroacetic acid (725 mL) and the mixture
stirred overnight under nitrogen, allowing the temperature to
rise back to ambient (caution, gas evolution!). Trifluoroacetic
acid was evaporated under reduced pressure and the residue
cooled in an ice bath. MeOH (1 L) was added carefully and
the mixture stirred until homogeneous. The solution was then
transferred to a 5 L three-necked flask fitted with a mechanical
stirrer, thermometer, and addition funnel and immersed in
an ice-water bath. NaOH (2.5 N) was added dropwise, keeping
the temperature of the mixture below 25 °C, until pH 11 was
reached (∼1 L). The reaction was stirred and the pH adjusted
to 11-12 by periodical addition of more 2.5 N NaOH, until it
remained constant for 1 h. Water (2.2 L) was added, and the
slightly turbid solution filtered to remove insoluble impurities
(mostly DCU). The filtrate was then diluted with water to a
volume of 5 L, and concentrated HCl was added dropwise
under vigorous stirring until a pH of 1 was reached (∼70 mL).
The resulting suspension was stirred for an additional hour
at room temperature and filtered, using water for rinses and
washings (4 × 2 L). The white solid was dried in air to constant
crystallize. Hexane (1.2 L) was added and the slurry stirred
overnight at room temperature. The white solid was collected
by filtration, washed with 2:1 TBME/hexane and hexane, and
dried in air to constant weight (347.6 g, 87% yield). The
material was 98.78% homogeneous (tR 15.12 min) as deter-
mined by reversed-phase HPLC (Supelcosil LC-ABZ, 4.6 × 150
mm, 5 µm particle size, 0-100% 0.1% TFA-CH3CN in 0.1%
TFA in 20 min then 100% 0.1% TFA in CH3CN for 5 min, 1
mL/min flow rate, UV detection at 215 nm), and contained
none of the P2 epimer as determined by co-injection with
analytical standards (see the Supporting Information for
preparation of epimeric P2 standards): mp 139.5-140.5 °C;
Rf 0.52 (5% MeOH/EtOAc); [R]25 - 30.8° (c 1.01, MeOH); IR
D
(film on NaCl) ν 3364, 2924, 1651, 1636 cm-1; 1H NMR (CDCl3,
mixture of rotamers) δ 8.60 (dq, J ) 5, 1 Hz, 0.1H), 8.56 (dq,
J ) 5, 1 Hz, 0.4H), 8.52 (dq, J ) 5, 1 Hz, 0.5H), 7.68-7.58 (m,
1H), 7.25-7.11 (m, 2H), 6.78 (d, J ) 8 Hz, 0.8H), 6.65 (d, J )
8.5 Hz, 0.1H), 6.60 (d, J ) 8 Hz, 0.1H), 5.29-5.20 (m, 2H),
4.41-4.22 (m, 2H), 4.11 (broad m, 0.7H), 4.01 (dd, J ) 17.5,
11 Hz, 0.1H, part of ABX), 3.92 (dd, J ) 17.5, 14 Hz, 0.1H,
part of ABX), 3.83-3.59 (m, 3H), 3.29-2.72 (m, 10H), 2.93 (s,
3H), 2.67-2.18 (m, 3H), 1.88 (m, 1H), 1.80-1.46 (m, 12H),
1.45-1.03 (m, 10H), 0.99-0.70 (m, 4H), 0.94, 0.93 (two d, J )
6.5 Hz, 3H total), 0.88, 0.87 (two d, J ) 6.5 Hz, 3H total); 13
C
NMR (CDCl3, mixture of rotamers) δ 175.0, 174.7 (2 C), 172.0,
171.9, 171.8, 171.7, 167.7, 167.6, 167.5, 158.5, 158.2, 157.9,
157.4, 149.7, 149.5, 148.4 (broad), 139.8, 139.7, 137.3 (broad),
136.9, 136.8, 124.0, 123.9, 123.8, 122.2, 122.0, 121.9, 121.8,
115.2, 114.9, 114.7, 77.8, 77.5, 77.4, 77.2, 69.7, 69.6, 55.4, 55.3,
53.9, 53.8, 50.0, 49.7, 49.4, 49.0, 48.8, 49.5, 48.4, 47.9, 47.8,
47.5, 47.4, 42.7, 42.6, 41.4, 41.3, 41.2, 40.8, 40.6, 38.9, 38.8,
38.7, 37.2, 37.0, 36.4, 36.3, 36.3, 36.2, 35.5, 35.4, 35.2, 35.0,
34.8, 34.6, 33.9, 33.8, 33.7 (2 C), 33.6, 33.5, 33.2, 32.7, 32.6,
31.0, 30.9, 30.8, 30.7, 26.9, 26.5, 26.3, 26.2 (2 C), 26.1, 25.8,
25.7, 24.6, 24.0, 21.8, 21.7; MS (ES-) m/z 687 (M - H). Anal.
Calcd for C38H61ClN4O5: C, 66.21; H, 8.92; N, 8.13. Found: C,
66.17; H, 9.20; N, 8.10.
weight (299.8 g, 99% yield): mp 187-188.5 °C; [R]25 -51.0°
D
1
(c 1.05, MeOH); IR (KBr) ν 3500-2300, 3254, 1730 cm-1; H
NMR (DMSO-d6) δ 7.82 (d, J ) 9 Hz, 1H), 5.35 (broad s, 1H),
5.21 (broad d, J ) 1 Hz, 1H), 4.67 (broad d, J ) 2 Hz, 1H),
4.49 (broad d, J ) 2.5 Hz, 1H), 4.09 (broad dt, J ) 9, 4 Hz,
1H), 3.05 (m, 2H), 2.91 (broad d, J ) 7.5 Hz, 1H), 2.63 (dd J
) 15, 7.5 Hz, 1H, part of ABX), 2.46 (dd, J ) 16.5, 8 Hz, 1H,
part of ABX), 2.42 (dd, J ) 14, 7 Hz, 1H, part of ABX), 2.28
(dd, J ) 16.5, 6 Hz, 1H, part of ABX), 1.76 (m, 1H), 1.70-1.53
(m, 6H), 1.48-1.38 (m, 1H), 1.37-1.27 (broad m, 1H), 1.24-
1.04 (m, 5H), 0.94-0.72 (m, 2H), 0.87 (d, J ) 6.5 Hz, 3H), 0.79
(d, J ) 6.5 Hz, 3H); 13C NMR (DMSO-d6) δ 173.9, 172.4, 139.2,
114.7, 76.5, 68.7, 46.7, 42.4, 40.8, 39.4, 38.6, 35.5, 33.5, 33.3,
32.0, 26.2, 25.8, 25.6, 24.1 (2 C), 21.4; MS (ES-) m/z 416 (M -
H). Anal. Calcd for C21H36ClNO5: C, 60.35; H, 8.68; N, 3.35.
Found: C, 60.46; H, 8.71; N, 3.37.
Cou p lin g of Acid 31 w ith Am in e 2. P r ep a r a tion of
Com p ou n d 6. Acid 31 (241.8 g, 578 mmol) was suspended in
dry THF (1.5 L) and the slurry cooled in an ice-water bath.
N-Methylmorpholine (127.2 mL, 1.160 mol) was added slowly
and the mixture stirred in ice for 30 min, until all solids
dissolved. Pivaloyl chloride (70.45 g, 584 mmol) in THF (100
mL + 25 mL rinse) was added dropwise (40 min), keeping the
temperature of the reaction mixture below 7 °C. After the
mixture was stirred for an additional 2 h at 0-5 °C, a mixture
of amine 210 (175.8 g, 607 mmol) and N-methylmorpholine
(76.3 mL, 691 mmol) in THF (100 mL + 25 mL rinse) was
added dropwise (30 min). After the resulting mixture was
stirred at 5 °C for 0.5 h, the ice bath was removed and the
mixture stirred overnight at room temperature. Water (2 L)
was then added and THF removed under reduced pressure.
The residue was partitioned with EtOAc (3 L) and 5 N NaOH
(250 mL). The organic layer was separated and the aqueous
phase extracted with additional EtOAc. The combined organic
phases were washed with 0.5 N NaOH and brine and dried
over MgSO4/activated charcoal. After filtration, EtOAc was
removed under reduced pressure, and the thick yellow residue
was redissolved in hot (60 °C) tert-butylmethyl ether (TBME,
2.4 L). The solution was allowed to cool slowly to room
temperature, under rapid stirring, causing compound 6 to
Con ver sion of Vin yl Ch lor id e 6 to Cr u d e BILA 2157
BS (1). Vinyl chloride 6 (340.4 g, 0.494 mol) was dissolved in
CH2Cl2 (2.00 L), and 2-methoxypropene (189 mL, 1.976 mol,
4 equiv) was added, followed by a catalytic amount of para-
toluenesulfonic acid monohydrate (2.35 g, 12.35 mmol). The
mixture was stirred for 64 h at room temperature, after which
time TLC showed complete conversion to acetonide 32 (Rf from
0.73 to 0.83 in 7.5% MeOH/CHCl3). Volatiles were removed
under reduced pressure and the residue coevaporated with
TBME (2 × 1 L) to yield 32 as a clear yellow oil.
The crude product from above was dissolved in TBME (1.7
L) and the solution cooled in an ice-water bath. Ice-cold water
(1.7 L) was added, followed by N-bromosuccinimide (92.33 g,
518.7 mmol, 1.05 equiv) in one portion. After the mixture was
stirred for 2.5 h at 0 °C, TLC indicated complete disappearance
of starting material (Rf from 0.83 to 0.73 in 7.5% MeOH/
CHCl3). Thiourea (45.12 g, 593 mmol, 1.2 equiv) was added at
0 °C in one portion and the mixture stirred for 2 h in the ice-
water bath. The reaction mixture was then diluted with cold
(0 °C) 0.2 M NaCl (5 L) and additional TBME (1 L). After
stirring, the organic layer was separated and the aqueous
phase washed again with TBME (4 × 2L). The aqueous phase
containing salts of compound 34 was then warmed to 65-70
°C and stirred for 2 h under a stream of nitrogen to affect
deprotection of the acetonide and removal of all traces of
organic solvents. After being cooled to room temperature, the
aqueous solution of crude BILA 2157 BS hydrochloride was
filtered through a 0.45 µm membrane. The filtrate (∼7 L) was
then added dropwise (20 mL/min) to vigorously stirred 0.5 M
NaOH (3 L, 1.5 mol, 3 equiv), causing precipitation of the free
base of BILA 2157 BS (final pH of aqueous phase was 5.8-
6.0). The precipitated product was collected by filtration,
washed with water, and dried in a stream of air to give 1 (302.8
g, 84% yield) as a light tan-colored solid. Analysis by reversed-
phase HPLC (Symmetry C8 column, 0.2 × 15 cm, 0.4 mL/min
flow rate, UV detection at 260 nM, 30-55% CH3CN in 50 mM
NaH2PO4 (pH 4.4) in 25 min gradient, then 55-80% in 10 min
then linear): tR 23.11 min (94.6% homogeneity).

6634 J . Org. Chem., Vol. 64, No. 18, 1999
Beaulieu et al.
P u r ifica tion of BILA 2157 BS (1). Crude 1 from above
(299.6 g, 412 mmol) was suspended in water (2 L), and
methanesulfonic acid (73.27 g, 762 mmol, 1.85 equiv) in water
(1 L) was added. NaCl (35.10 g) was added and the solution
washed with CH2Cl2 (3 × 1.5 L). The aqueous layer was then
stirred for 3 h under a stream of nitrogen to remove any last
traces of solvent and the solution filtered through a 0.45 µm
membrane. The solution was then added dropwise (3.5 h, 15
mL/min) to vigorously stirred 1 N NaOH (770 mL) diluted with
water to a volume of 1.5 L (0.513 N final concentration). After
the suspension was stirred overnight at room temperature,
the precipitated product was collected by filtration, washed
with water, and dried in air to constant weight (273.6 g, 91%
recovery, 96.2% homogeneity by HPLC; see below for condi-
tions).
39.0, 37.2, 37.0, 36.5, 36.45, 36.25, 36.2, 36.0, 35.8, 35.0, 34.9,
34.8, 34.3, 34.2, 33.9, 33.8, 33.7, 33.65, 33.55, 33.2, 32.6, 30.9,
30.85, 30.8, 30.7, 26.5, 26.4, 26.35, 26.2, 26.15, 26.1, 25.85, 25.8,
25.75, 24.7, 24.0, 21.9; MS (ES+) m/z 727 (MH+). Anal. Calcd
for C39H62N6O5S (corrected for 2.85% w/w water content as
determined by Karl Fisher analysis): C, 62.58; H, 8.67; N,
11.23. Found: C, 62.24; H, 8.73; N, 11.01. HPLC homogeneity
(Symmetry C8, 0.2 × 15 cm, 30-55% CH3CN in 50 mM NaH2-
PO4 at pH 4.4 in 25 min then 55-80% CH3CN in 50 mM NaH2-
PO4 at pH 4.4 in 10 min, 0.4 mL/min flow rate, UV detection
at 260 nm): tR 22.8 min (97.74%). P2 epimer of BILA 2157
BS: tR 24.05 min (<0.01%).
Ackn owledgm en t. We are grateful to Colette Bouch-
er, Sylvain Bordeleau, Michael Little, Nancy Shore, and
Serge Valois for analytical support. We are also thankful
to Drs. Karl Grozinger and J ohn Landi (Boehringer
Ingelheim Pharmaceuticals, Inc.) for supplies of ami-
nodiol 4 and Dr. Michel Simard of the Universite´ de
Montre´al for the X-ray structure determination. Finally,
we would like to thank Dr. Bruno Simoneau for useful
comments and Dr. Paul C. Anderson for his continued
support and encouragement.
The procedure was repeated starting with 1 from above (261
g, 0.359 mol) using 1.95 equiv of methanesulfonic acid (67.27
g, 0.700 mol) for salt formation. Neutralization was carried
out using NaOH (30.54 g, 0.763 mol) in water (1.5 L). Following
filtration of the precipitated free base, the product was
collected and washed twice by stirring (30 min) with water (4
L). Finally, BILA 2157 BS was dried in air to give an off-white
solid: mp 85-93 °C (lit.10 mp 92-97 °C); Rf 0.25 (5% MeOH/
CHCl3); [R]25 -26.8° (c 1.08, MeOH); [R]25 -80.6° (c 1.08,
D
365
MeOH) (lit.10 [R]25 -26.0° (c 1.02, MeOH)); IR (NaCl film) ν
D
3700-2800, 1640 cm-1 1H NMR (DMSO-d6 and CDCl3,
;
mixture of rotamers) identical to that previously reported;6,10
13C NMR (CDCl3, mixture of rotamers) δ 176.1, 176.0, 175.9,
172.2, 169.0, 168.95, 168.9, 168.8, 168.0, 167.8, 167.7, 167.6,
158.9, 158.7, 158.0, 157.6, 149.75, 149.5, 149.3, 149.2, 149.15,
136.9, 136.7, 136.65, 136.6, 123.8, 123.7, 123.6, 123.5, 122.2,
121.9, 121.7, 121.5, 103.8, 103.7, 103.6, 78.3, 78.2, 78.1, 77.2,
69.8, 55.4, 55.2, 53.8, 53.7, 50.0, 49.7, 49.0, 48.9, 48.6, 48.0,
47.7, 47.4, 47.2, 42.7, 42.65, 42.55, 42.50, 42.45, 39.15, 39.1,
Su p p or tin g In for m a tion Ava ila ble: HPLC chromato-
grams for compounds (R)- and (S)-15. Experimental procedures
for the preparation and characterization of isomeric analytical
standards: (R)-19, P2 epimers of compounds 1, 6, 27, 30, and
31 and diol isomers of compounds 6, 30, and 31. This material
is available free of charge via the Internet at http://pubs.acs.org.
J O990321X