Regioselective enzymatic aminoacylation of lobucavir to give an intermediate for lobucavir prodrug

Article abstract of DOI:10.1016/S0968-0896(00)00209-1

Synthesis of lobucavir prodrug, L-valine, [(1S,2R,3R)-3-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobucavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1R,2R,4S)-2-(2-amino-6-oxo-1H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester (3, 82.5% yield), was obtained by selective hydrolysis of N,N'-bis[(phenylmethoxy)carbonyl]bis[L-valine], O,O'-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M, and L-valine, [(1R,2R,4S)-2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (4, 87% yield) was obtained by hydrolysis of bis[L-valine], O,O'-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester, dihydrochloride (2), with lipase from Candida cylindracea. The final intermediate for lobucavir prodrug, N-[(phenylmethoxy)carbonyl]-L-valine, [(1S,2R,4R)-3-(2-amino-6-oxo-1H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester (5), could be obtained by transesterification of lobucavir using ChiroCLEC(TM) BL (61% yield), or more selectively by using immobilized lipase from Pseudomonas cepacia (84% yield). Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00209-1

Bioorganic & Medicinal Chemistry 8 (2000) 2681±2687
Regioselective Enzymatic Aminoacylation of Lobucavir to
Give an Intermediate for Lobucavir Prodrug
Ronald L. Hanson, a,* Zhongping Shi, b David B. Brzozowski, a Amit Banerjee, a
Thomas P. Kissick, b Janak Singh, b Annie J. Pullockaran, b Je€rey T. North, b
Junying Fan, b Je€rey Howell, a Susan C. Durand, a Michael A. Montana, a
David R. Kronenthal, a Richard H. Mueller b and Ramesh N. Patel a
aDepartment of Process Research and Development, Bristol-Myers Squibb Pharmaceutical Research Institute,
One Squibb Drive, New Brunswick, NJ 08903, USA
bDepartment of Process Research and Development, Bristol-Myers Squibb Pharmaceutical Research Institute,
PO Box 4000, Princeton, NJ 08543, USA
Received 24 March 2000; accepted 6 July 2000
AbstractÐSynthesis of lobucavir prodrug, L-valine, [(1S,2R,3R)-3-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-2-(hydroxymethyl)
cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobu-
cavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic
reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1R,2R,4S)-2-(2-amino-6-oxo-1H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl
ester (3, 82.5% yield), was obtained by selective hydrolysis of N,N0-bis[(phenylmethoxy)carbonyl]bis[L-valine], O,O0-[(1S,2R,3R)-3-
(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M, and L-valine, [(1R,2R,4S)-2-(2-amino-1,6-dihydro-
6-oxo-9H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (4, 87% yield) was obtained by hydrolysis of
bis[L-valine], O,O0-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester, dihydrochloride (2), with lipase
from Candida cylindracea. The ®nal intermediate for lobucavir prodrug, N-[(phenylmethoxy)carbonyl]-L-valine, [(1S,2R,4R)-3-(2-
amino-6-oxo-1H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester (5), could be obtained by transesteri®cation of lobucavir
using ChiroCLECTM BL (61% yield), or more selectively by using immobilized lipase from Pseudomonas cepacia (84% yield).
# 2000 Elsevier Science Ltd. All rights reserved.
Introduction
50-butyrylation using vinyl butyrate or tri¯uoroethyl
butyrate with subtilisins.3 Wong and co-workers devel-
oped mutant subtilisins with several amino acid sub-
stitutions that gave better stability in DMF and were
used successfully for 50-O-acetylation of nucleosides.4,5
Lipases were also shown to carry out regioselective
acylations of pyrimidine nucleosides in organic solvent
using vinyl acetate or acid anhydrides.6,7 Moris and
Gotor reported that oxime esters were useful acyl
donors for regioselective acylation of nucleosides by
lipases8,9 and extended this technique to regioselective
aminoacylation using oxime aminoacyl esters prepared
from p-nitrophenyl esters of Z-gly or Z-or Boc b-ala.10
Lobucavir (BMS 180194, Scheme 2) is a cyclobutyl
guanine nucleoside analogue recently under develop-
ment as an antiviral agent for treatment of herpes vir-
uses and hepatitis B.1 A prodrug form in which one of
the two hydroxyls is coupled to valine, BMS 233866
(Scheme 3), has also been considered for development.
Regioselective aminoacylation by valine is dicult to
achieve by chemical procedures, but appeared to be
suitable for an enzymatic approach. Riva et al. were the
®rst to demonstrate enzymatic regioselective acylation
of nucleosides, using trichloroethyl butyrate and sub-
tilisin in N,N-dimethylformamide (DMF).2 Singh et al.
reported that using pyridine as a solvent instead of
DMF gave higher yields and better regioselectivity for
Regioselective enzymatic hydrolysis of mutiply-acylated
nucleosides has also been used as an approach to selec-
tive acylation. Selective 50-O-deacetylation of tri-O-
acetylribonucleosides by subtilisin or porcine pancreatic
lipase was reported by Singh et al.11 Katagiri et al.
found that lipase MY deacylated the 50-O and lipase xiii
*Corresponding author. Tel.: +1-732-519-2797; fax: +1-732-519-
2797; e-mail: ronald.hanson@bms.com
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00209-1

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R. L. Hanson et al. / Bioorg. Med. Chem. 8 (2000) 2681±2687
Scheme 1. Regioselective hydrolysis of diesters 1 and 2.
Scheme 2. Transesteri®cation of Z-valine p-nitrophenyl ester with lobucavir.
(Pseudomonas sp.) deacylated the 30-O of carbocyclic
3050-di-O-acyloxetanocin.12
lipase from Candida cylindracea, which gave 87 M%
yield of 3 on a 2 g scale in phosphate bu€er as described
in Experimental.
Regioselective acylation of only one of the 2 hydroxyls
of lobucavir with N-protected L-valine is a problem that
we have approached by chemical aminoacylation of
both hydroxyl groups followed by selective enzymatic
hydrolysis and also by selective enzymatic aminoacyla-
tion.
Coupling
Enzyme and solvent screening. Initial e€orts to couple
lobucavir to Z-valine by transesteri®cation focused on
using lipases that converted 1 to 3, or 2 to 4 in the
hydrolysis direction (lipases M, C. cylindracea, and
others). If these enzymes selected the same hydroxyl site
in the coupling reaction, lobucavir would be converted
to the desired product (5, Scheme 2). These lipases did
not give any coupled products, but ChiroCLECTM BL
(cross-linked crystals of subtilisin) gave 5. Undesired
side products were also produced in smaller amounts (3
and 1). The rate and yield for the coupling reaction were
very dependent on the choice of solvent. The best yield
for the reaction was obtained using 70% acetone/30%
DMF as solvent (Fig. 1). Addition of 0.01 or 0.1%
water slightly and 1.0% water strongly decreased the
rate and yield of the coupling reaction. Other solvents
Results
Hydrolysis
Screening lipases for regioselective hydrolysis of diester
1 revealed several that gave 3, but none that gave the
desired product 5. On a 50 g scale, lipase M in 90%
toluene/10% bu€er as described in Experimental gave 3
in 75.2% yield. Lipases were also screened for hydroly-
sis of 2 to BMS 233866. Again, selectivity was for the
undesired isomer 4. The most selective enzyme was

R. L. Hanson et al. / Bioorg. Med. Chem. 8 (2000) 2681±2687
2683
Scheme 3. Transesteri®cation of Boc-valine p-nitrophenyl ester with lobucavir and subsequent deprotection.
Figure 1. E€ect of DMF concentration on yield after 2 days. The
reaction contained 10 mg/mL lobucavir, 50 mg/mL (3.56 equiv)
Z-valine p-nitrophenyl ester, and 15 mg/mL ChiroCLECTM BL.
Figure 2. Kinetics of tranesteri®cation of Z-valine p-nitrophenyl ester
with lobucavir using ChiroCLECTM BL. The reaction contained
10 mg/mL lobucavir, 50 mg/mL Z-valine p-nitrophenyl ester, and
15 mg/mL ChiroCLECTM BL in 70% acetone/30% DMF. Initial
lobucavir concentrations are dicult to assay because of the low
solubility in the reaction solvent.
giving lower yields and/or lower regioselectivity were
tetrahydrofuran, DMF, acetone, acetonitrile, methylene
chloride, methyl t-butyl ether, methyl ethyl ketone,
methylisobutyl ketone, ethyl acetate, toluene, isooctane,
pyridine, dimethyl sulfoxide, dioxane, t-butanol, and t-
amyl alcohol.
The time course of a 7 g batch is shown in Figure 2. The
yield for this reaction was 8.30 g, 63.1 M% 5 (desired
product). Undesired products were 3 (1.02 g, 7.8 M%), 1
(2.55 g, 13.2 M%), and unreacted BMS 180194

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R. L. Hanson et al. / Bioorg. Med. Chem. 8 (2000) 2681±2687
(2.1 M%). The reaction was scaled up to give 607 g of 5
(53.8 M%) from a 600 g input of lobucavir and 4.5 kg of
5 (54.8 M%) from a 4.4 kg input of lobucavir (Table 2).
Table 2. Scaleup of transesteri®cation of Z-valine p-nitrophenyl ester
with lobucavir
Lobucavir Desired product, 5
input, g
Other
g
Molar% yield
61.4
Molar% yield
Reusability
7
8.07*
BMS 180194
3
1
BMS 180194
2.4
ChiroCLECTM BL was used three times without a
noticeable loss of activity, but yields dropped on the
fourth and ®fth uses. Unreacted Z-valine p-nitrophenyl
ester, recovered after isolation of 5, was used in a sec-
ond coupling reaction to give 5 in 58.5% yield.
7.6
12.6
0.4
600
4400
607
53.8
54.8
3
7.2
1
BMS 180194
23.0
2.6
4531
3
1
7.2
11.7
Other esters with ChiroCLECTM BL
*Mean of 3 reactions.
Z-valine methyl ester gave only about 15% of the yield
obtained with the p-nitrophenyl ester, and the reaction
was much slower. In 20 mg scale experiments, Z-valine
tri¯uoroethyl ester gave a somewhat better yield than
the p-nitrophenyl ester. Four equivalents Z-valine
N-hydroxysuccinimide ester gave a 50% yield after 4
days, and reaction was a little slower than with the p-
nitrophenyl ester. N-Boc-valine p-nitrophenyl ester gave
a signi®cant improvement in yield to 76 M% (Table 1)
and reaction rate. The amount of enzyme required to
complete the reaction in about 52 h was decreased by
half when the Boc-ester was substituted for the Z-ester.
(13%). Rate, yield, and regioselectivity of the transesteri®-
cation of Z-valine p-nitrophenyl ester with lobucavir
using ChiroCLECTM BL were strongly a€ected by sol-
vent. 70% Acetone/30% DMF gave the highest rate
and yield of 5 of any of the solvents tested.
Rate and yield in the aminoacylation of lobucavir by
ChiroCLECTM BL were also strongly a€ected by the
leaving group and N-protecting groups on the valine
ester. When several Z-L-valine esters were compared,
p-nitrophenyl=tri¯uoroethyl>N-hydroxysuccinimide
>>methyl ester in rate and yield for the reaction.
When Boc-L-valine p-nitrophenyl ester was used, fol-
lowed by deprotection of analytical samples, a 76%
yield of BMS 233866 was achieved using 1/2 the amount
of ChiroCLECTM BL required for the reaction with the
Z-valine p-nitrophenyl ester. Undesired side products
were 4 (8.3%) and 2 (11.5%).
Transesteri®cation by Pseudomonas cepacia lipase
In a 20 mg scale reaction, ChiroCLEC-PC using 7 equiv
Z-valine tri¯uoroethyl ester gave an 84.6% yield of 5
with no 3 and only 1.8% 1 (Fig. 3). Under the same
conditions, Amano lipase PS30 immobilized on Accurel
polypropylene gave an 81.4% yield of 5 with no 3 and
only 0.7% 1.
Wong and co-workers developed mutant subtilisins with
several amino acid substitutions that gave better stabil-
ity in DMF and were used successfully for 50-O-acetyla-
tion of nucleosides.4,5 The cross-linked enzyme crystals
of subtilisin are an alternative for increased stability in
organic solvents and were reused for four more reac-
tions in acetone/DMF.
Discussion
Either hydroxyl group of lobucavir could be selectively
aminoacylated with valine by using enzymatic reactions.
Monoester 3 (82.5% yield) was obtained by selective
hydrolysis of 1 with lipase M, and 4 (87% yield) was
obtained by hydrolysis of 2 with lipase from C. cylin-
dracea. However, these two products were not the
desired intermediates for lobucavir prodrug. Chiro-
CLECTM BL (cross-linked crystals of subtilisin) cata-
lyzed a regioselective aminoacylation of lobucavir using
Z-L-valine p-nitrophenyl ester as acyl donor to give 5 in
62% yield. Undesired side products were 3 (7.8%) and 1
ChiroCLECTM PC (cross-linked crystals of P. cepacia
lipase) or Amano lipase PS30 (also P. cepacia lipase)
immobilized on Accurel polypropylene gave the highest
yield and regioselectivity. Z-valine tri¯uoroethyl ester
was transesteri®ed with lobucavir to give 5 in 84% yield,
with no 3 and only 0.4% 1. With this lipase Z-valine p-
nitrophenyl ester was not an e€ective substrate. Moris
Table 1. Yields in transesteri®cation reactions with lobucavir
Enzyme
Ester
Desired product
Molar% yield
63.1
Other
Molar% yield
CLECTM BL
Z-valine p-nitrophenyl ester
5
BMS 180194
3
2.1
7.8
1
BMS 180194
13.2
7.1
8.3
11.5
0.3
CLECTM BL
CLECTM PC
Boc-valine p-nitrophenyl ester
BMS 233866
76.0
84.6
4
2
BMS 180194
Z-valine tri¯uoroethyl ester
5
3
1
0.0
1.9

R. L. Hanson et al. / Bioorg. Med. Chem. 8 (2000) 2681±2687
2685
phase. The combined toluene extracts were pooled and
concentrated at 40 ꢀC (ꢁ23 mmHg) to give 40.4 g oily
solid containing 20.8 g 3.
Isolation and puri®cation of 3
A warm solution of crude 3 (40.2 g containing 19.96 g 3)
in ethyl acetate (700 mL) was washed with water
(1Â150 mL and 1Â50 mL) by stirring at 45 ꢀC (bath).
The separated organic phase was then treated with
additional water (50 mL) at 45 ꢀC (bath). The mixture
was slowly cooled to room temperature with stirring
and stirred at room temperature for 4 h. A white pre-
cipitate formed as the organic phase was cooled to room
temperature, the solid was collected by ®ltration, washed
with ethyl acetate:hexane (10:90), air-dried and dried in
vacuo (18 h) to provide the desired 3 as a white solid
(18.85 g, 98.4 HPLC area%, 94% yield): mp 99.5 ꢀC, [a]D
0.19.3ꢀ (c 1.01, MeOH). 1H NMR (270 MHz, DMSO-
d6) d 7.83 (s, 1H), 7.62 (d, 1H, J=7.6 Hz), 7.22±7.43 (m,
5H), 6.35 (s, 2H), 5.03 (s, 2H), 4.67 (t, 1H, J=5.4 Hz),
4.38±4.53 (m, 1H), 4.17 (d, 2H, J=5.3 Hz), 3.88±3.83 (t,
1H, J=7.0 Hz), 3.53 (m, 2H), 2.92 (m, 1H), 2.36 (m,
1H), 2.00±2.23 (m, 2H), 1.87 (m, 1H), 0.78 (d, 6H,
J=6.4 Hz); 13C NMR (100 MHz, DMSO-d6) d 171.5,
156.6, 156.2, 153.0, 150.8, 136.7, 135.5, 128.1 (2 C),
127.6 (2 C), 127.5, 116.6, 65.3, 65.1, 62.9, 59.4, 47.0,
43.8, 33.1, 29.3 (2 C), 18.7, 17.7. IR (KBr) 3423, 3337,
2691, 1700, 1628, 1535, 1095 cm 1. Anal. calcd for
Figure 3. Kinetics of transesteri®cation of Z-valine tri¯uoroethyl ester
with lobucavir using ChiroCLECTM PC. The reaction contained
10 mg/mL lobucavir, 89.4 mg/mL (7.12 equiv) Z-valine tri¯uoroethyl
ester, and 15 mg/mL ChiroCLECTM PC in 80% t-butyl-methyl ether/
20% DMF. The ®rst sample was taken after 24 h.
and Gotor also noted that p-nitrophenyl esters of N-
protected amino acids were not e€ective aminoacylating
agents when using P. cepacia lipase and other lipases.10
Conclusion
.
The ®nal intermediate (5) for lobucavir prodrug could
be obtained by transesteri®cation of lobucavir using
ChiroCLECTM BL (61% yield), or more selectively by
using immobilized lipase from P. cepacia (84% yield).
Coupling of Z-valine or valine to the other hydroxyl
was achieved by chemical aminoacylation of both
hydroxyls, followed by regioselective enzymatic hydro-
lysis using lipase M from Mucor javanicus or lipase from
C. cylindracea, respectively.
C24H30N6O6 1.0 H2O: C, 55.78; H, 6.25; N, 16.27.
Found: C, 55.47; H, 6.10; N, 16.01.
Hydrolysis of diester 2
Diester 2 (2.0 g) and 20 g lipase from C. cylindracea were
stirred in 800 mL 0.1 M phosphate bu€er, pH 7.0, at
28 ꢀC for 18 h. Reaction product concentrations deter-
mined by HPLC were 1.62 mg/mL 4 (87M%), 0.006 mg/
mL BMS 233866, 0.2 mg/mL BMS 180194, and
0.025 mg/mL remaining diester 2.
Experimental
Coupling reactions
Hydrolysis of 1
Analytical scale coupling reactions were carried out on
a heated block in 2 mL capped vials mixed with a mag-
netic stirring bar. For a preparative batch, lobucavir
(7.00 g, 26.39 mmol), N-CBZ-L-valine p-nitrophenyl
ester (35 g, 93.98 mmol, 3.56 equiv), and 10.5 g Chiro-
CLEC BL were added to a jacketed 1-L three-necked
round-bottom ¯ask equipped with an air-driven stirrer
and a condenser maintained at 5 ꢀC by a circulating
water bath. 490 mL acetone followed by 210 mL DMF
were added, a stopper was put in the third neck of the
¯ask and the mixture was stirred fast enough to keep the
enzyme and lobucavir suspended. The jacket tempera-
ture was raised to 45 ꢀC with a second circulating water
bath, and the reaction was monitored by taking 0.1 mL
samples for analysis by HPLC. Other reaction condi-
tions are described in the ®gure legends. The immobi-
lized enzyme was removed by ®ltration at the end of the
reaction, and acetone was removed with a rotary evap-
orator at 40 ꢀC. The recovered immobilized enzyme was
stored at 4 ꢀC for reuse. A 600 g batch was carried out in
Diester 1 (50.0 g, 68.32 mmol) was dissolved in 2.5 L
toluene by stirring at 50±55 ꢀC, then added to a 15-L
jacketed reactor equipped with an air-driven stirrer and
a condenser kept at 4±8 ꢀC. The reactor was heated to
40 ꢀC with a circulator. Another 7.5 L toluene was
added to the reactor. The stirrer was turned to full
power and 250 g lipase MAP-10 was added to give a ®ne
suspension of the enzyme. One litre of 0.1 M potassium
phosphate bu€er, pH 7.0, was added, and the reaction
was continued until HPLC analysis indicated that the
concentration of 1 had decreased to 0.12 g/L. Reactions
were stopped after 71 h. HPLC analysis showed 3
(28.38 g, 83.3 M%), 1 (1.32 g, 2.6 M%), 5 (0.06 g,
0.2 M%), and BMS 180194 (0.13 g, 0.7 M%). After
completion of the reaction, the whole solution was passed
through a coalescing ®lter to break the emulsion with
the resulting toluene layer separated. The remaining
small amount of emulsion was further extracted with
toluene followed by separation of the resulting organic

2686
R. L. Hanson et al. / Bioorg. Med. Chem. 8 (2000) 2681±2687
a 100 L reactor under nitrogen, and a 4.4 kg batch was
carried out in a pilot plant reactor.
HPLC analysis
For analysis of hydrolysis reactions, samples were evap-
orated to dryness under a nitrogen stream, then dis-
solved in 75% acetonitrile/25% potassium dihydrogen
phosphate (0.68%) and 1-heptanesulfonic acid, sodium
salt (0.1%), in water. For coupling reactions with Z-valine
esters, 0.1 mL samples were evaporated to dryness
under a nitrogen stream at 45 ꢀC. The residue was taken
up in 2 mL 50% acetonitrile/50% water containing
0.4% tri¯uoroacetic acid. To this sample was added
1 mL 50% acetonitrile, 50% water containing 0.05 M
KH2PO4 and 0.01% heptanesulfonic acid, sodium salt.
After mixing and sonicating, 1 mL acetonitrile was added
and the sample was mixed and sonicated again. For
coupling reactions with Boc-valine p-nitrophenyl ester,
samples were dried, treated for 30 min at room tem-
perature with tri¯uoroacetic acid, dried again, then dis-
solved as outlined for the Z-valine ester samples.
Isolation and puri®cation of 5
A concentrated reaction mixture (678.0 g) from the
selective enzymatic acylation of BMS 180194 (20.0 g)
with p-nitrophenyl ester of CBZ-L-valine (200 g) in
DMF±acetone was concentrated to remove most of the
DMF at 40 ꢀC (ꢁ1 mm Hg) to provide a crude product
(318 g, containing ꢁ100 g DMF). One portion of this
crude (130.0 g) was treated with ethyl acetate (200 mL)
and water (80 mL). Crystalline 5 seeds were added to the
mixture, which was then stirred at 25 ꢀC for 0.5 h and
then at 65 ꢀC for 4 h. The mixture was cooled to 25 ꢀC
for 3 h and then cooled to 0 ꢀC (ice bath) for 20 min. The
crystals were collected by ®ltration, washed with water
(20 mL) and ethyl acetate (15 mL), and dried under
vacuum to give 8.1 g (46% yield, 98 HPLC area%) of 5
with 98 AP. The crude 5 was further puri®ed by stirring
in ethyl acetate (100 mL) and water (30 mL) at 65 ꢀC
(bath) for 3 h and then cooling to ambient temperature.
Filtration and washing with ethyl acetate (15 mL) fol-
lowed by drying under vacuum provided 7.6 g (43%) of
pure 5 as a white solid: mp 205.0±206.0 ꢀC. [a]D 0.6ꢀ (c
1.02, MeOH). 1H NMR (270 MHz, DMSO-d6) d 7.91 (s,
1H), 7.72 (d, 1H, J=7.6 Hz), 7.20±7.45 (m, 5H), 6.43 (s,
2H), 5.05 (s, 2H), 4.70 (t, 1H, J=4.7 Hz), 4.50 (m, 1H),
4.12±4.31 (m, 2H), 3.96 (t, 1H, J=7.4 Hz), 3.49 (m, 2H),
3.37 (s, 1H, OH), 2.77 (m, 1H), 2.40 (m, 1H), 2.26 (m,
1H), 1.95±2.20 (m, 2H), 0.91 (d, 6H, J=6.5 Hz); 13C
NMR (68 MHz, DMSO-d6) d 173.1, 158.1, 157.7,
154.5, 152.3, 138.1, 137.3, 129.6 (2 C), 129.1 (3 C),
118.0, 68.0, 66.8, 62.0, 61.1, 49.2, 47.5, 31.3, 30.9, 30.6,
Samples were analyzed with a YMC, Inc., ODS-A, C-18,
5 m, 4.6Â150 mm column. The mobile phase was a gra-
dient of A: potassium dihydrogen phosphate (0.68%)
and 1-heptanesulfonic acid, sodium salt (0.1%), in water
and B: acetonitrile (0 min 5%, 30 min 14%, 50 min 14%,
70 min 50%, 100 min 50%, 105 min 5%, 110 min 5%).
Flow rate was 1 mL/min, detection was at 254 nm, tem-
perature was ambient, and injection volume was 20 mL.
Retention times (min) were: BMS 180194, 7.2; 4, 25.5;
BMS 233866, 31.8; 2, 55.5; Z-valine, 58.5; 3, 63.4; 5,
64.9; Z-valine methyl ester, 72.9; 1, 76.2; Z-valine-p-
nitrophenyl ester, 88.2.
Materials
20.3, 19.5. IR (KBr) 3600±3000, 1740, 1694, 1624,
1
1601, 1534, 1362, 1028 cm
.
Anal. calcd for
Commercial materials were obtained from the following
sources: Z-L-valine and Z-valine tri¯uoroethyl esters,
Chem-Impex International, Boc-L-valine p-nitrophenyl
ester, Bachem; Z-valine N-hydroxysuccinimide ester,
Nova; ChiroCLECTM BL and ChiroCLECTM PC,
Altus Biologics; M-10 (Mucor javanicus) lipase, Amano;
C. cylindracea lipase, Biocatalysts; lipase PS30 (P.
cepacia) from Amano was immobilized by adsorption
on Accurel polypropylene as described previously.13
C24H30N6O6: C, 57.76; H, 6.07; N, 16.84. Found: C,
57.67; H, 5.79; N, 16.62.
Recovery of excess p-nitrophenyl ester of CBZ-L-valine
The mother liquor from the ®rst crystallization was
concentrated and treated with ethyl acetate:hexane (2:5,
500 mL) at 50 ꢀC for 0.5 h. The mixture was then cooled
to 0 ꢀC (ice bath, 1 h). A small amount of a brown oil
was present. The supernatant was decanted to a 1 L
¯ask. Ethyl acetate (100 mL) was added to the super-
natant, which was then washed with aqueous K3PO4
solution (10%, 3Â150 mL) and NaH2PO4 (std. 100 mL)
and dried (Na2SO4). The solid (Na2SO4) was removed
by ®ltration. Concentration of the ®ltrate provided an
oil which was dissolved in ethyl acetate (70 mL)/hexane
(300 mL) at 40 ꢀC (oil bath). The solution was seeded
and additional hexane (500 mL) was added while stir-
ring and slowly cooling to 0 ꢀC. The crystalline solid
was collected by ®ltration and washed with hexane
(50 mL) to give 49.0 g of p-nitrophenyl ester of CBZ-L-
valine (>99.5 AP) as a white solid: mp 65.0±66.0 ꢀC
(identical to an authentic sample). The mother liquor
was concentrated and an additional 4.5 g of p-nitrophe-
nyl ester of CBZ-L-valine (>99.5 AP) was obtained in
the second crop by crystallization from ethyl acetate
(20 mL) and hexane (280 mL).
Preparation of diester 1
Cbz-L-valine (3.02 g, 5.0 mmol) and EDC (3.26 g,
17.0 mmol) were added to a suspension of BMS 180194
(monohydrate, 1.417 g, 5.0 mmol) in MeCN (20 mL)
under an argon atmosphere. After 2.5 h, the reaction
was quenched with AcOH (0.3 g, 5.0 mmol) and the
solvent was removed under vacuum. The residue was
treated with ethyl acetate (60 mL) and water (20 mL).
The organic phase was washed with water (3Â20 mL),
1 M aq H3PO4 (3Â15 mL), brine (10 mL), aq NaHCO3
(3Â15 mL) and brine. During these washings, 2 mL of
brine was added each time after shaking to facilitate the
separation of the layers. The organic solution was dried
(MgSO4), ®ltered, and concentrated, and the residue
was dried under vacuum to provide 4.07 g (98.3 AP) of
crude product. Crystallization of the crude product
from EtOAc:TBME:heptane (2:2:1, 25 mL) gave 2.99 g

R. L. Hanson et al. / Bioorg. Med. Chem. 8 (2000) 2681±2687
2687
(82%, 99.4 AP) of 1 as a white solid. 1H NMR
(400 MHz, DMSO-d6) d 10.71 (s, 1H), 7.91 (s, 1H), 7.74
(d, 1H, J=8.1 Hz), 7.63 (d, 1H, J=8.1 Hz), 7.23±7.45
(m, 10H), 6.44 (s, 2H), 4.97±5.10 (m, 4H), 4.52 (m, 1H),
4.26 (d, 1H, J=5.1 Hz), 4.20 (d, 1H, J=5.1 Hz), 4.08±
4.25 (m, 2H), 3.98 (dd, 1H, J=6.8 Hz, 7.7 Hz), 3.89 (dd,
1H, J=6.0 Hz, 8.0 Hz), 3.00 (m, 1H), 2.42 (m, 1H), 2.30
(m, 1H), 2.23 (m, 1H), 2.06 (m, 1H), 1.89 (m, 1H), 0.91
(d, 6H, J=6.4 Hz), 0.78 (d, 6H, J=5.0 Hz); 13C NMR
(68 MHz, DMSO-d6) d 172.1, 172.0, 157.2, 156.7 (2 C),
153.7, 151.4, 137.2 (2 C), 136.2, 128.6 (4 C), 128.1 (6 C),
117.1, 66.7, 65.9, 65.1, 60.2, 60.0, 59.9, 47.3, 45.0, 30.3,
30.1, 29.9, 29.8, 19.3, 19.2, 18.5, 18.2. IR (KBr) 3600±
3000, 1701, 1626, 1595, 1532, 1028 cm 1. Anal. calcd for
1.00 (d, 3H, J=7.0 Hz), 0.96 (d, 3H, J=7.0 Hz), 0.86 (d,
3H, J=7.0 Hz), 0.84 (d, 3H, J=7.0 Hz); 13C NMR
(68 MHz, DMSO-d6) d 168.7 (2 C), 156.2, 154.1, 150.8,
136.3, 115.0, 67.4, 65.6, 57.4 (2 C), 47.2, 44.2, 29.5, 29.3
(2 C), 29.2, 18.7, 18.3, 17.6 (2 C). IR (KBr) 3430, 1744,
1
.
1686, 1630, 1595 cm . Anal. calcd for C21H33N7O5 2
.
HCl 1.0 H2O: C, 44.55; H, 6.75; N, 17.09; Cl, 14.05.
Found: C, 44.48; H, 6.72; N, 16.95; Cl, 14.13.
References and Notes
1. Ireland, C.; Leeson, P. A.; Castaner, J. Drugs Future 1997,
22, 359.
2. Riva, S.; Chopineau, J.; Kieboom, A. P. G.; Klibanov, A.
M. J. Am. Chem. Soc. 1988, 110, 584.
.
C37H45N7O9 0.35 H2O: C, 60.20; H, 6.24; N, 13.29.
Found: C, 59.89; H, 6.17; N, 13.10.
3. Singh, H. K.; Cote, G. L.; Had®eld, T. M. Tetrahedron
Lett. 1994, 35, 1353.
Preparation of 2
4. Wong, C.-H.; Chen, S.-T.; Hennen, W. J.; Bibbs, J. A.;
Wang, Y.-F.; Liu, J. L.-C.; Pantoliano, M. W.; Whitlow, M.;
Bryan, P. N. J. Am. Chem. Soc. 1990, 112, 945.
5. Zhong, Z.; Liu, J. L.-C.; Dinterman, L. M.; Finkelman, M.
A. J.; Mueller, W. T.; Rollence, M. L.; Whitlow, M.; Wong,
C.-H. J. Am. Chem. Soc. 1991, 113, 683.
6. Uemura, A.; Nozaki, K.; Yamashita, J.; Yasumoto, M.
Tetrahedron Lett. 1989, 30, 3817.
7. Nozaki, K.; Uemura, A.; Yamashita, J.; Yasumoto, M.
Tetrahedron Lett. 1990, 31, 7327.
8. Moris, M.; Gotor, V. J. Org. Chem. 1993, 58, 653.
9. Moris, M.; Gotor, V. Tetrahedron 1993, 44, 10089.
10. Moris, M.; Gotor, V. Tetrahedron 1994, 50, 6927.
11. Singh, H. K.; Cote, G. L.; Sikorski, R. S. Tetrahedron
Lett. 1993, 34, 5201.
12. Katagiri, N.; Morishita, Y.; Yamaguchi, M. Tetrahedron
Lett. 1998, 39, 2613.
13. Patel, R. N.; Banerjee, A.; Ko, R. Y.; Howell, J. M.; Li,
W.-S.; Comezoglu, F. T.; Partyka, R. A.; Szarka, L. Bio-
technol. Appl. Biochem. 1994, 20, 23.
A solution of 1 (45.54 g, 62.3 mmol) in MeOH (500 mL)
and 10% Pd/C (4.55 g) were added to a 2-L Parr
hydrogenation bottle under an Ar atmosphere. Con-
centrated HCl (10.3 mL, 125 mmol) was added and the
mixture was hydrogenated at 25 psi for 4 h (reaction was
complete by TLC and HPLC). The reaction mixture
was sparged with N2 for 1 h, ®ltered through Celite, and
washed with MeOH. Concentration of the ®ltrate pro-
vided the crude product as a foam. The foam was tritu-
rated with acetone (500 mL) and then stirred for 2 h.
The product was collected by ®ltration, washed with
acetone, and dried under vacuum to give 32.7 g (97.1%,
ꢁ100 AP) of 2 as a white solid: mp 192.5 ꢀC. [a]D
+15.6ꢀ (c 1.0, MeOH). 1H NMR (270 MHz, DMSO-d6)
d 8.66 (br-s, 7H), 8.25 (s, 1H), 6.77 (s, 2H), 4.63 (m, 1H),
4.20±4.46 (m, 4H), 3.93 (br-s, 1H), 3.79 (br-s, 1H), 3.10
(m, 1H), 2.28±2.53 (m, 3H), 2.22 (m, 1H), 2.06 (m, 1H),