Structure activity studies of the serine-AIB dipeptide domain in 2,3-dihydroisothiazole based growth hormone secretagogues

Article abstract of DOI:10.1016/j.bmc.2005.07.070

A series of growth hormone secretagogues (GHSs) based on 2,3-dihydroisothiazole has been synthesized in the search for a potential treatment of growth hormone deficiency or frailty in the elderly. This paper describes the evaluation of the SAR of the benzyl-d-Ser-aminoisobutyric acid dipeptide fragment. Introduction of substituents in the peptide backbone and in the phenyl ring has been investigated, as well as replacements for the benzyl group and for the AIB residue. A number of modifications resulted in enhanced potency over the parent benzyl-d-Ser-AIB derivative.

Full text of DOI:10.1016/j.bmc.2005.07.070

Bioorganic & Medicinal Chemistry 13 (2005) 6748–6762
Structure activity studies of the serine-AIB dipeptide domain
in 2,3-dihydroisothiazole based growth hormone secretagogues
Britta Evers,a,* Gerd Ruehter,a Martina Berg,a Jeffrey A. Dodge,b Dirk Hankotius,a
Ulrike Hary,a Louis N. Jungheim,b Hans-Juergen Mest,a Eva-Maria Martin de la Nava,a
Michael Mohr,a Brian S. Muehl,b Soenke Petersen,a Birgit Sommer,a Grit Riedel-Herold,a
Mark J. Tebbe,b Kenneth J. Thrasherb and Silke Voelkersa
aLilly Forschung GmbH, Division of Eli Lilly Research Laboratories, Essener Bogen 7, 22419 Hamburg, Germany
bLilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA
Received 21 April 2005; revised 18 July 2005; accepted 21 July 2005
Available online 10 October 2005
Abstract—A series of growth hormone secretagogues (GHSs) based on 2,3-dihydroisothiazole has been synthesized in the search for
a potential treatment of growth hormone deficiency or frailty in the elderly. This paper describes the evaluation of the SAR of the
benzyl-D-Ser-aminoisobutyric acid dipeptide fragment. Introduction of substituents in the peptide backbone and in the phenyl ring
has been investigated, as well as replacements for the benzyl group and for the AIB residue. A number of modifications resulted in
enhanced potency over the parent benzyl-D-Ser-AIB derivative.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
formed by O-benzyl-D-serine and aminoisobutyric acid
(AIB), or carbon-analogues such as those found in LY
444711.5,6 Examples include MK-06777 and CP-4243918
shown in Figure 1. The structure-function studies of ghre-
lin have been summarized9 and suggest that the dipeptide
corresponds tothe N-terminus ofthe GHRPsand ghrelin.
Most research groups have made minimal changes to this
structural motif, while focussing their attention on modi-
fications of other part of the molecule.
Growth hormone secretagogues (GHSs) have been
widely studied during the past several years as an alter-
native to (recombinant) growth hormone for the treat-
ment of growth hormone deficiency and frailty in the
elderly. These compounds release endogenous growth
hormone through interaction with the GHS receptor
(GHS-R1a), a G-protein-coupled receptor, occurring
predominantly in the pituitary gland and in the hypo-
thalamus. The first GHSs discovered were oligopeptides,
called growth hormone releasing peptides (GHRPs), like
the hexapeptide GHRP-6.1 Later, small orally bioavail-
able peptidomimetic GHSs were also identified.2 Inter-
estingly, it was only in 1999 that the endogenous
GHS-receptor ligand, ghrelin, was discovered.3 This
28-amino acid peptide contains a unique octanoyl-serine
residue and is secreted in the stomach.
In this paper, we present a part of the results of our work
on a series of 2,3-dihydroisothiazole GHSs.10–12 Based on
compound 1 that has shown potent growth hormone
releasing activity in a rat pituitary cell assay, we have ex-
plored both the steric and electronic requirements of the
dipeptide moiety in this series. One approach was to sys-
tematically introduce methyl groups into different posi-
tions of the serine. In addition, we varied the chain
length of the linker, introduced substituents into the ben-
zyloxy group and replaced it by heterocycles.
Many of the small molecule GHSs described in the litera-
ture, some of which have been advanced to clinical inves-
tigations,4 contain as a common feature a dipeptide
2. Chemistry
To study the effect of substituted benzyl serine or of het-
erocyclic benzyl replacements, a series of D-serine ana-
logues was prepared. The building blocks were
Keywords: Growth hormone; Growth hormone secretagogue.
*
Corresponding author. Tel.: +49 40 527 24 566; fax: +49 40 527 24
601; e-mail: Evers_Britta@lilly.com
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.070

B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
6749
H
H
N
N
H
N
H
N
O
NH2
NH2
O
NH2
O
NH2
O
O
O
O
O
O
N
NH
O
O
N
N
N
N
N
N
S
N
NH
Cl
O
O
S
O
N
O
O
O
O
LY 444711
MK-0677
CP-424391
1
Figure 1. Growth hormone secretagogues with a common dipeptide structural motif.
obtained by alkylation of N-Boc-D-serine (2) with
substituted benzyl or heteroarylmethyl halides (Scheme
1). Subsequent coupling and deprotection steps with
the amine 410 and with Boc-AIB (7) yielded the products
9a–z and 11a–j.
To study the effect of N-methylserine, the alanine ana-
logues 22 and 23 were prepared following the route out-
lined in Scheme 3. Boc-protected benzyl-D-serine (19)
was converted into the oxazolidinone 20 by refluxing
with paraformaldehyde and p-TosOH. Subsequent
reductive cleavage with Et3SiH and TFA,14 followed
by reprotection with Boc, led to the N-Me-serine inter-
mediate 21 in good yield.
N-Boc-D-2-amino-5-phenyl-pentanoic acid (15a) and its
fluorinated analogues 15b–d were prepared as shown in
Scheme 2 analogous to known procedures. Diethyl ace-
tamidomalonate (12) was alkylated with (substituted)
1-bromo-3-phenylpropanes, and the products were sub-
sequently decarboxylated under basic conditions. Acyl-
ase-mediated deacylation13 of the racemic mixture
afforded the D-N-acetyl amino acids 14a–d selectively.
They were deacylated, the amino groups were Boc-pro-
tected, and the resulting esters were cleaved to afford
15a–d, which were then coupled with amine 4 under stan-
dard conditions. Deprotection and subsequent coupling
to Boc-AIB according to the methods in Scheme 1, fol-
lowed by final Boc removal, provided the products 16a–d.
Intermediate 6 and its 2,6-difluorobenzyl analogue 6s
were applied in coupling reactions with a number of
Boc-protected amino acids to afford compounds with
replacements for the aminoisobutyric acid such as 2-flu-
oromethyl-3-fluoro-alanine15 (24), 1-amino-cyclopro-
pane-carboxylic acid (25), a-methyl-serine (26 and 27)
and the alanine derivatives 28 and 29.
3. Results and discussion
The N-terminal structure of ghrelin, the natural ligand
for the GHS receptor, begins with a Gly-Ser-Ser tripep-
tide, in which the serine in the third position is esterified
with octanoic acid. Simple overlay of the structures of
In a route analogous to that used to access the D-serine
series, N-Boc-benzyl-D-allo-threonine (17) was convert-
ed to derivative 18, as depicted in Scheme 3.
NHEt
H
NHR1
N
O
O
NHR1
NH
X
HO
S
O
X
NHBoc
NHBoc
O
N
O
O
O
O
O
N
Cl
X
7
4
O
OH
NH
O
NH
3a-z
S
b
b
S
O
O
O
Cl
Cl
a
R1 = Boc
R1 = H
5a-z
6a-z
R1 = Boc
8a-z
NHBoc
OH
c
d
HO
9a-z R1 = H
O
2
H
N
R2
O
NH2
a
O
O
N
R2
(4)
1.
2.
, b, c
NHBoc
OH
O
NH
(7) , b, d
S
O
O
O
or
for 10i:
FMOC-AIB, e, f
Cl
10a-i
11a-j
Scheme 1. Synthesis of D-serine derivatives. Reagents and conditions: (a) NaH, DMF, rt, overnight; (b) DCC, HOBt, CH2Cl2, rt; (c) TFA, CH2Cl2,
rt; (d) HCl, EtOH, rt; (e) TBTU, NEt3, rt; (f) DMF, EtNH2, rt.

6750
B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
X
O
O
X
OEt
X
X
H
N
O
Br
NHBoc
OH
HN
HN
OEt
O
d, e, b
b, c
O
O
OH
O
EtO
EtO
O
a
O
14a-d
15a-d
12
13a-d
H
N
NH2
X
O
(4)
1.
2.
, f, g
O
N
(7) , f, h
NH
O
a X = H
S
b X = 4-F
O
Cl
c X = 3,5-F2
d X = 2,6-F2
16a-d
Scheme 2. Synthesis of D-2-amino-5-phenyl-pentanoic acid analogues. Reagents and conditions: (a) Na, EtOH, reflux; (b) NaOH, reflux; (c) acylase,
CoCl2, KOH; (d) HCl, EtOH, reflux; (e) (Boc)2O, NEt3, THF, rt; (f) DCC, HOBt, CH2Cl2, rt; (g) TFA, CH2Cl2, rt; (h) HCl, EtOH, rt.
H
N
O
NH2
O
NHBoc
(4)
1.
2.
, a, b
O
O
N
(7) , a, c
O
OH
NH
O
17
S
O
Cl
18
N
N
O
NH2
O
Boc
N
O
NHBoc
OH
NBoc
OH
(4)
1.
2.
d
a, b
O
e, f
O
O
O
a, c
O
O
O
NH
S
19
20
21
O
HO
O
Cl
NHBoc
O
22 (D-Ala)
23 (L-Ala)
R
X
H
N
NH2
F
F
R
O
O
X
24 2,6-F2
X
a, c
N-Boc-amino acid
O
N
NH2
O
N
O
NH
O
NH
O
25
H
NH2
S
S
O
O
O
Cl
Cl
26
27
28
29
H
H
H
H
D-alpha-Me-Ser
L-alpha-Me-Ser
D-Ala
6
(X = H)
6s (X = 2,6-F2)
L-Ala
Scheme 3. Synthesis of methyl analogues of 1 and AIB replacements. Reagents and conditions: (a) DCC, HOBt, CH2Cl2, rt; (b) TFA, CH2Cl2, rt;
(c) HCl, EtOH, rt; (d) paraformaldehyde, p-TosOH, toluene, reflux; (e) Et3SiH, TFA, CH2Cl2; (f) (Boc)2O, NEt3, THF, rt.
benzylserine derived GHSs with that of ghrelin suggests
that the benzylserine moiety of the GHSs binds in the
same region of the receptor as the octanoyl chain. This
has also been demonstrated by an octanoyl-serine deriva-
tive of MK-0677 that retained some activity in the
functional assay.16 We explored the steric and electronic

B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
6751
requirements of binding to this pocket in a series of ben-
zyl-serine-2,3-dihydro-isothiazole-based GHSs, which
contained primarily hydrophobic residues in the dipep-
tide. All compounds were tested for their ability to
promote secretion of growth hormone on isolated rat
pituitary cells.
logues 9t and 9w–9y demonstrated activity in the sub-
nanomolar range. We assume that this effect can be
explained by stabilisation of a favourable conformation
of the aromatic residue through hindered rotation of the
benzylic C–C bond. Therefore, the observed gain in in vi-
tro activity is more pronounced with chlorine substituents
compared to the smaller fluorine atoms.
We first studied the influence of substituents on the phenyl
ring of the benzyl serine moiety (Table 1). We found that
substitution in the para-position reduced the level of in vi-
tro activity, relative to substitution in the meta- or ortho-
position (compare 9a with 9b or 9c; 9i with 9j; 9e with 9k).
Generally, polar substituents, such as trifluoromethoxy
(9f), carboxamide (9m), or the 4-cyano derivative 9i, led
to decreased levels of in vitro activity. An exception is
the 2-cyano analogue 9j (EC50 = 1.9 nM). We also pre-
pared various ortho-substituted benzyl derivatives. Intro-
duction of substituents in only one of the ortho-positions
caused little or no increase in in vitro activity compared
with the parent compound 1. However, when a substitu-
ent like halogen was incorporated into both ortho-posi-
tions, the resulting compounds showed a significant
increase in the level of in vitro activity when compared
with compound 1. In fact, the 2,6-dihalogenated ana-
The phenyl ring of the O-benzyl-D-serine moiety was re-
placed with a series of heterocyclic groups. The results
are shown in Table 2. Whereas all the nitrogen-contain-
ing heterocycles tested showed moderate in vitro activity
(EC50 = 22 to >100 nM), the hydrophobic 3- and 2-thi-
enyl derivatives 11g and 11h proved to have comparable
in vitro activity (EC50 values 3.4 and 3.6 nM, respective-
ly) to the parent compound 1.
Replacement of the ether linker in the benzylserine with its
carbon analogue, 2-amino-5-phenyl-pentanoic acid,
Table 2. Heterocyclic replacements for the benzyl group
H
R
N
NH2
O
O
N
Table 1. Activities of substituted benzyl derivatives in the pituitary cell
assay
NH
S
O
O
H
N
Cl
O
NH2
X
O
Compound
R
EC50 (nM)
3.5
SEM
0.7
O
N
O
1
NH
S
O
O
Cl
N
11a
11b
11c
11d
11e
11f
11g
11h
38
43
28a
O
Compound
X
EC50 (nM)
SEM
1
H
4-F
3.5
12.1
3.1
3.1
8.9
38
0.7
5.4
9a
9b
9c
9d
9e
9f
N
O
3a
3-F
2-F
0.9
1.5
4-Cl
1.2
18a
4-CF3
4-OCF3
4-Cl-2-F
2-Cl-4-F
4-CN
2-CN
2-CF3
2-CH3
O
54
22
>83
8.2
8.1
>71
1.9
8.3
8.8
55
N
N
9g
9h
9i
3.5
2.5
O
>100
22
9j
0.5
2.4
3.2
24a
0.7
0.4
2.4
0.8
1.1
0.5
O
9k
9l
N
9m
9n
9o
9p
9q
9r
9s
9t
2-CONH2
2,3-Cl2
2,3-F2
O
8
2.0
2.7
8.0
2.0
7.0
1.1
0.1
18
S
2,4-F2
2,5-F2
N
O
47
17
3,5-F2
2,6-F2
S
2,6-Cl2
2,4,5-F3
2,3,5-F3
2,3,6-F3
2-Cl-3,6-F2
2-Cl-6-F
0.05a
O
O
9u
9v
9w
9x
9y
9z
6.8
0.3
0.07
0.5
3.4
3.6
0.4
1.5
1.3
0.3
0.5
<0.1
1.4
S
S
2,6-F2-3-CH3
0.4
a n = 2.
a n = 2.

6752
B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
resulted in a modest improvement of in vitro activity (Ta-
ble 3, entries 1 and 16a). This trend of increased activity in
the carbon series was also shown with a number of fluori-
nated carbon analogues. Indeed, in all cases the carbon
analogues showed at least 2-fold more in vitro activity
than their ether counterparts (Table 3). The greatest in-
crease in in vitro activity, approximately 10-fold, was seen
in the case of 2,6-difluoro substitution (compare com-
pounds 9s and 16d). Compound 16d stimulated GH secre-
tion in the pituitary cell with an EC50 of 0.1 nM.
already optimal. Extension of the chain by one atom
via incorporation of benzyl-D-homoserine (30), as well
as shortening the linker by one methylene group as in
the D-homophenylalanine derivative (31), resulted in a
reduction in the level of in vitro activity (Table 3).
Finally, we investigated how additional substituents
were tolerated along the dipeptide chain, using the meth-
yl group as an example (Table 4). We found that an
Table 4. Methyl derivatives of 1 and AIB variations
In addition, we studied the effect of the chain length of
the linker on the activity and found that the three-atom
linker between phenyl and asymmetric carbon was
N
R1 =
NH
Cl
S
O
O
Table 3. Linker variations
H
R
N
Cpd.
EC50 (nM)
3.5
SEM
0.73
NH2
O
H
N
O
N
O
NH2
1
O
O
R1
NH
H
N
S
NH2
NH2
NH2
O
O
O
O
11i
11j
18
22
23
24
25
26
27
28
0.9
0.3
0.12
0.19
O
Cl
O
O
O
O
O
O
R1
Compound
R
EC50 (nM)
3.5
SEM
0.73
H
N
O
1
R1
H
N
>100
>96
3.4
O
O
9a
12.1
7.0
5.4
1.1
0.5
R1
F
F
N
O
O
NH2
NH2
O
O
9r
O
O
R1
F
F
N
2.5
9s
1.1
O
O
R1
F
F
F
F
H
N
78
13a
O
O
NH2
30
>56
1.1
O
O
F
O
O
R1
H
N
NH2
>100
16a
16b
16c
16d
31
0.5
0.7
R1
OH
H
N
O
NH2
0.7
0.1
2.6
O
2.3
O
F
F
R1
OH
H
N
O
O
NH2
20.3
>100
4.4
O
O
3.7
1.3
O
O
R1
F
H
N
F
NH2
0.1
0.09
R1
x
F
H
N
29
1.2
O
NH2
58
38a
O
O
R1
a n = 2.
a n = 2.

B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
6753
additional methyl group in the benzylserine was only
tolerated if it were distal to the a-carbon. Compared
to the unsubstituted compound 1, the two isomers 11i
and 11j with the methyl group in the benzylic position
had significantly improved in vitro activity with EC50
values of 0.9 and 0.3 nM, respectively. In contrast, for
the D-allo-threonine derivative 18 a reduction of in vitro
activity was observed.
analogues a more favourable orientation of the aromatic
residue is stabilised. This becomes especially apparent for
the chlorine derivatives, whereas the conformation of 9s
with the smaller fluorine atoms can be considered as being
more similar to the unsubstituted 1. Such an effect could
also explain the improved activity of the a-methylbenzyl
analogues 11i and 11j.
We demonstrated that the chain length of the linker in
benzylserine was optimal, and that seemingly small
changes in the AIB moiety decreased activity, with the
exception of the hydroxylated compound 26, which dis-
played a 5-fold improvement in EC50 over the corre-
sponding AIB analogue 1.
To study the effect of N-methylation, we prepared the N-
Me-Ala analogues 22 and 23. Comparison with their NH-
Ala analogues 28 and 29 shows that L-alanine (23 and 29)
provides comparable in vitro activity with parent 1,
whereas the D-alanine analogues 22 and 28 reduced the
level of activity in the pituitary cell assay. N-Methylation
in the serine is well-tolerated for good AIB replacements.
A detailed summary of our results describing the SAR
of the dihydroisothiazole moiety will be published
elsewhere.
The sensitivity of the GHS activity to minor modifica-
tions within the AIB moiety was illustrated by com-
pounds 24 and 25, in which fluorination of the methyl
groups or their replacement by cyclopropane, respec-
tively, resulted in a reduction in the level of in vitro
activity, including an example with the 2,6-difluoro-
phenyl ring as in compound 24. Interestingly, however,
selective hydroxylation of one of the AIB-methyl groups
results in a 5-fold increase in activity versus parent 1 (R-
isomer 26), whereas hydroxylation of the other methyl
group results in a 6-fold reduction (S-isomer 27). The
preference of the R-isomer is in agreement with results
from other series.17
5. Experimental
5.1. General chemistry methods
All reagents and solvents were obtained from commercial
sources and were used without further purification. H
1
NMR spectra were recorded on a Bruker Avance
300 MHz or on a Varian Inova 500 MHz in CDCl3 or
DMSO-d6 as solvent using the deuterium lock signal as
internal standard. Chemical shift data are given as d
(ppm) values. Low-resolution mass spectra were obtained
a Perkin-Elmer Sciex API 150 MCA using electrospray
ionisation (ESI). Preparative chromatographic separa-
tions were performed using flash chromatography with
silica gel (Millipore, 60A, 35–70 lm).
4. Conclusions
The SAR of the benzyl-D-Ser-AIB dipeptide fragment in a
series of dihydroisothiazole-based GHSs has been evalu-
ated thoroughly. Starting from GHS 1 with an EC50 of
3.5 nM, a number of modifications led to additional
improvement of the in vitro activity. In agreement with
the assumption that the benzylserine moiety occupies
the same region at the GHS receptor as the octanoyl side
chain of the N-terminus of ghrelin, nonpolar replace-
ments for the benzyl residue were well-tolerated. Whereas
heterocycles, such as, pyridines and thiazoles, decreased
the in vitro activity, the thienyl analogues 11g and 11h
were found to have comparable in vitro activity to 1,
and also the a-methylbenzyl analogues 11i and 11j are
good examples of how hydrophobic groups can contrib-
ute to efficient ligand binding at the receptor. The two iso-
mers 11i and 11j show a 3-fold difference in activity. The
absolute stereochemistry and the influence of larger
groups in that position remain to be evaluated.
5.2. Method A: serine alkylation
N-Boc-D-serine (2) (1 g, 4.9 mmol) was dissolved in
DMF and cooled to 5 ꢁC, NaH (60% in mineral oil,
0.49 g, 12.25 mmol) was added and the mixture was stir-
red for 30 min. The substituted benzyl halide or the cor-
responding heteroarylmethyl bromide (1.05 equiv) was
added and the mixture was stirred overnight at rt. After
evaporation, the residue was dissolved in water (50 ml),
extracted with t-butylmethylether, acidified with 10%
citric acid and extracted with ethyl acetate. The ethyl
acetate layer was dried (Na2SO4) and evaporated. Chro-
matography on silica with a CH2Cl2–EtOH gradient
yielded the product.
5.3. Method B: coupling with amine 4 and deprotection
The greatest gain in in vitro activity was achieved through
the introduction of 2,6-dihalogenated benzyl groups as in
9t, 9w and 9y. This gain cannot be attributed to electronic
effects only, because other derivatives with electron-defi-
cient phenyl rings, such as, 9g, 9h and 9p, do not display
the same boost in activity. Instead, steric effects seem to
play a role, which is demonstrated by a reduction in the
level of in vitro activity with the introduction of para-sub-
stituents. We assume that through hindered rotation of
the benzylic C–C bond in case of the 2,6-disubstituted
5.3.1. 2-(R)-2-Amino-3-benzyloxy-N-[5-(4-chlorophenyl)-
3,3-dimethyl-1,1-dioxo-2,3-dihydro-isothiazol-4-ylmethyl]-
N-ethyl-propionamide
(6,
X = H).
[5-(4-Chloro
phenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydro-isothiazol-
4- ylmethyl]-ethylamine10 (4, 500 mg, 1.59 mmol) was sus-
pended in isopropylacetate (10 ml). Water (5 ml), DCC
(361 mg, 1.75 mmol), HOBT (237 mg, 1.75 mmol) and
N-Boc-O-benzyl-D-serine (517 mg, 1.75 mmol) were
added and the mixture was stirred overnight at room tem-
perature. The mixture was filtered, rinsed with isopropy-

6754
B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
lacetate, and the aqueous phase was separated. The organ-
ic layer was washed with citric acid (0.1 M) and satd NaH-
CO3, dried over Na2SO4 and concentrated. The residue
was dissolved in CH2Cl2 (10 ml) and TFA (7.5 ml) was
added at 0 ꢁC. The solution was allowed to warm to room
temperature and stirred overnight. After evaporation, the
residue was dissolved in CH2Cl2, washed with satd NaH-
CO3 and satd NaCl solutions, dried over Na2SO4 and con-
centrated to yield 6 (440 mg, 56%) as an amorphous solid.
1H NMR (DMSO-d6) d 7.56 (m, 2H), 7.43 (m, 2H), 7.38–
7.24 (m, 5H), 4.41 (m, 3H), 4.19 (d, 1H, J = 5.8 Hz), 3.55
(m, 1H), 3.23 (m, 2H), 3.04 (m, 2H), 1.40 (m, 6H), 0.82
(t, 3H). MS m/z 492 (M++Na).
(d, 1H, J = 8.3 Hz), 4.61 (s, 2H), 4.48 (m, 1H), 3.96 (m,
1H), 3.74 (m, 1H), 1.45 (s, 9H). MS m/z 312 (MÀÀ1).
5.4.5. N-tert-Butoxycarbonylamino-O-(4-chlorobenzyl)-D-
serine (3d). The title compound was prepared from 2 and
4-chlorobenzyl bromide by Method A (yield = 43%). H
1
NMR (CDCl3) d 9.61 (br s, 1H), 7.29 (m, 2H), 7.22 (m,
2H), 5.41 (d, 1H, J = 8.3 Hz), 4.48 (m, 3H), 3.90 (m,
1H), 3.71 (dd, 1H, J1 = 3.4 Hz J2 = 9.4 Hz), 1.45 (s,
9H). MS m/z 328 (MÀÀ1).
5.4.6. N-tert-Butoxycarbonylamino-O-(4-trifluoromethyl-
benzyl)-D-serine (3e). The title compound was prepared
from 2 and 4-trifluoromethylbenzyl bromide by Method
A (yield = 66%). 1H NMR (DMSO-d6) d 12.6 (br s,
1H), 7.70 (m, 2H), 7.54 (m, 2H), 7.02 (d, 1H,
J = 8.3 Hz), 4.59 (m, 2H), 4.21 (m, 1H), 3.70 (m, 2H),
1.37 (s, 9H). MS m/z 386 (M++Na).
5.4. Method C: coupling with Boc-AIB and deprotection
5.4.1. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-phe-
nylmethoxypropionic
acid
N-(5-(4-chlorophenyl)-3,
3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-
N-ethylamide hydrochloride (1). 2-(R)-2-Amino-3-ben-
zyloxy-N-[5-(4-chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,
3-dihydro-isothiazol-4-ylmethyl]-N-ethyl-propionamide (6,
440 mg, 0.89 mmol) was suspended in isopropylacetate
(6 ml). Water (6 ml), DCC (202 mg, 0.98 mmol), HOBT
(132 mg, 0.98 mmol) and N-Boc-a-amino-isobutyric
acid (7, 200 mg, 0.98 mmol) were added and the mixture
was stirred overnight at room temperature. The mixture
was filtered, rinsed with isopropylacetate, and the aque-
ous phase was separated. The organic layer was washed
with citric acid (0.1 M) and satd NaHCO3, dried over
Na2SO4 and concentrated. The residue was purified by
chromatography on silica gel (CH2Cl2/EtOH 98:2).
The product was dissolved in 10% HCl in EtOH (4 ml)
and stirred overnight at room temperature. The mixture
was poured into diethyl ether (400 ml), stirred for 1 h
and the precipitate was filtered off and dried at 50 ꢁC un-
der vacuum to yield 1 as a white solid (320 mg, 59%). 1H
NMR (DMSO-d6) d 7.51 (d, 2H, J = 8.5 Hz), 7.43 (d,
2H, J = 8.5 Hz), 7.34–7.24 (m, 5H), 4.66 (m, 1H), 4.53
(d, 1H, J = 16.2 Hz), 4.41 (s, 2H), 4.05 (d, 1H,
J = 16.2 Hz), 3.34 (m, 1H), 3.05 (m, 3H), 1.45 (s, 3H),
1.43 (s, 3H), 1.39 (s, 6H), 0.92 (t, 3H, J = 6.9 Hz). MS
m/z 577 (MH+).
5.4.7. N-tert-Butoxycarbonylamino-O-(4-trifluorometh-
oxybenzyl)-D-serine (3f). The title compound was pre-
pared from 2 and 4-trifluoromethoxybenzyl bromide
by Method A (yield = 54%). 1H NMR (DMSO-d6) d
12.7 (br s, 1H), 7.44 (m, 2H), 7.32 (m, 2H), 7.00 (d,
1H, J = 8.3 Hz), 4.45 (m, 2H), 4.19 (m, 1H), 3.68 (m,
2H), 1.37 (s, 9H). MS m/z 402 (M++Na).
5.4.8. N-tert-Butoxycarbonylamino-O-(4-chloro-2-fluoro-
benzyl)-D-serine (3g). The title compound was prepared
from 2 and 4-chloro-2-fluorobenzyl bromide by Method
A (yield = 90%). 1H NMR (DMSO-d6) d 12.8 (br s,
1H), 7.58–7.40 (m, 2H), 7.23 (m, 1H), 6.98 (d, 1H,
J = 8.3 Hz), 4.51 (m, 2H), 4.19 (m, 1H), 3.74 (m, 2H),
1.37 (s, 9H). MS m/z 370 (M++Na).
5.4.9. N-tert-Butoxycarbonylamino-O-(2-chloro-4-fluoro-
benzyl)-D-serine (3h). The title compound was prepared
from 2 and 2-chloro-4-fluorobenzyl bromide by Method
A (yield = 39%). 1H NMR (DMSO-d6) d 12.8 (br s,
1H), 7.58–7.40 (m, 2H), 7.22 (m, 1H), 6.98 (d, 1H,
J = 8.3 Hz), 4.53 (m, 2H), 4.21 (m, 1H), 3.74 (m, 2H),
1.37 (s, 9H). MS m/z 370 (M++Na).
5.4.10. N-tert-Butoxycarbonylamino-O-(4-cyanobenzyl)-
D-serine (3i). The title compound was prepared from
5.4.2. N-tert-Butoxycarbonylamino-O-(4-fluorobenzyl)-D-
serine (3a). The title compound was prepared from 2 and
4-fluorobenzyl bromide by Method A (yield = 42%). H
NMR (CDCl3) d 8.85 (br s, 1H), 7.29 (m, 2H), 7.32 (dd,
2H, J1 = 8.5 Hz, J2 = 8.7 Hz), 5.44 (d, 1H, J = 8.3 Hz,
NH), 4.49 (s, 2H), 4.47 (m, 1H), 3.92 (m, 1H), 3.71
(m, 1H), 1.45 (s, 9H). MS m/z 312 (MÀÀ1).
2
and 4-cyanobenzyl bromide by Method
A
1
1
(yield = 99%). H NMR (CDCl3) d 7.47–7.37 (m, 2H),
7.33–7.24 (m, 2H), 4.64 (s, 2H), 4.50 (m, 1H), 4.03 (m,
1H), 3.79 (dd, 1H, J1 = 3.6 Hz, J2 = 9.3 Hz), 1.45 (s,
9H). MS m/z 319 (MÀÀ1).
5.4.11. N-tert-Butoxycarbonylamino-O-(2-cyanobenzyl)-D-
serine (3j). The title compound was prepared from 2 and
2-cyanobenzyl bromide by Method A (yield = 77%). H
5.4.3. N-tert-Butoxycarbonylamino-O-(3-fluorobenzyl)-D-
serine (3b). The title compound was prepared from 2 and
3-fluorobenzyl bromide by Method A (yield = 58%). H
1
1
NMR (CDCl3) d 7.67–7.48 (m, 2H), 7.38 (m, 1H), 5.50
(m, 1H), 4.72 (s, 2H), 4.52 (m, 1H), 4.03 (m, 1H), 3.83
(dd, 1H, J1 = 2.0 Hz, J2 = 9.09 Hz), 3.77 (dd, 1H,
J1 = 3.4 Hz, J2 = 9.5 Hz), 1.45 (s, 9H). MS m/z 319
(MÀÀ1).
NMR (CDCl3) d 8.89 (br s, 1H), 7.27 (m, 1H), 7.08–6.91
(m, 3H), 5.51 (d, 1H, 7.3 Hz), 4.51 (m, 3H), 3.93 (m,
1H), 3.73 (m, 1H), 1.45 (s, 9H). MS m/z 312 (MÀÀ1).
5.4.4. N-tert-Butoxycarbonylamino-O-(2-fluorobenzyl)-D-
serine (3c). The title compound was prepared from 2 and
2-fluorobenzyl bromide by Method A (yield = 39%). H
5.4.12. N-tert-Butoxycarbonylamino-O-(2-trifluorometh-
ylbenzyl)-D-serine (3k). The title compound was prepared
from 2 and 2-trifluoromethylbenzyl bromide by Method
1
NMR (CDCl3) d 8.84 (br s, 1H), 7.40–6.68 (m, 4H), 5.42

B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
6755
1
A (yield = 59%). H NMR (CDCl3) d 8.59 (br s, 1H),
5.4.19. N-tert-Butoxycarbonylamino-O-(3,5-difluoroben-
zyl)-D-serine (3r). The title compound was prepared
from 2 and 3,5-difluorobenzyl bromide by Method A
(yield = 57%). 1H NMR (CDCl3) d 9.75 (br s, 1H),
6.88–6.65 (m, 3H), 5.44 (d, 1H, J = 8.3 Hz), 4.52 (m,
3H), 3.95 (m, 1H), 3.75 (dd, 1H, J1 = 3.4 Hz,
J2 = 9.3 Hz), 1.45 (s, 9H). MS m/z 330 (MÀÀ1).
7.66–7.48 (m, 3H), 7.36 (m, 1H), 5.45 (d, 1H,
J = 8.3 Hz), 4.72 (s, 2H), 4.53 (m, 1H), 4.00 (m, 1H),
3.78 (dd, 1H, J1 = 3.4 Hz, J2 = 9.5 Hz), 1.45 (s, 9H).
MS m/z 362 (MÀÀ1).
5.4.13. N-tert-Butoxycarbonylamino-O-(2-methylbenzyl)-
D-serine (3l). The title compound was prepared from 2
and 2-methylbenzyl bromide by Method
A
5.4.20. N-tert-Butoxycarbonylamino-O-(2,6-difluoroben-
zyl)-D-serine (3s). The title compound was prepared
from 2 and 2,6-difluorobenzyl bromide by Method A
(yield = 75%). 1H NMR (CDCl3) d 8.15 (br s, 1H),
7.27 (m, 1H), 6.93–6.82 (m, 2H), 5.37 (d, 1H,
J = 8.3 Hz), 4.63 (s, 2H), 4.44 (m, 1H), 3.94 (m, 1H),
3.74 (m, 1H), 1.45 (s, 9H). MS m/z 330 (MÀÀ1).
(yield = 64%). 1H NMR (DMSO-d6) d 7.37–7.10 (m,
4H), 6.90 (d, 1H, J = 8.3 Hz), 4.47 (s, 2H), 4.19 (m,
1H), 3.67 (m, 2H), 2.25 (s, 3H), 1.38 (s, 9H). MS m/z
332 (M++Na).
5.4.14. N-tert-Butoxycarbonylamino-O-(2-carboxamido-
benzyl)-D-serine (3m). Compound 2 was alkylated with
2-cyanobenzyl bromide by Method A (yield = 97%).
Subsequently, sodium perborate tetrahydrate (1.44 g,
9.4 mmol) was dissolved in water (10 ml), the nitrile
(1 g, 3.12 mmol) and MeOH (10 ml) were added and stir-
red for 72 h. MeOH was evaporated and the residue was
extracted with CHCl3, the organic layer was dried
(Na2SO4) and concentrated. Chromatography on silica
(CH2Cl2/MeOH 10:1) yielded the title compound in
5.4.21. N-tert-Butoxycarbonylamino-O-(2,6-dichloroben-
zyl)-D-serine (3t). The title compound was prepared
from 2 and 2,6-dichlorobenzyl bromide by Method A
1
(yield = 69%). H NMR (CDCl3) d 7.33–7.15 (m, 3H),
5.40 (d, 1H, J = 8.5 Hz), 4.82 (s, 2H), 4.43 (m, 1H),
3.98 (m, 1H), 3.77 (dd, 1H, J1 = 3.6 Hz, J2 = 9.3 Hz),
1.45 (s, 9H). MS m/z 386 (M++Na).
1
47% yield. H NMR (CDCl3) d 7.78–7.27 (m, 4H), 5.60
5.4.22. N-tert-Butoxycarbonylamino-O-(2,4,5-trifluoro-
benzyl)-D-serine (3u). The title compound was pre-
pared from 2 and 2,4,5-trifluorobenzyl bromide by
Method A (yield = 90%). 1H NMR (DMSO-d6) d
7.60–7.48 (m, 2H), 7.03 (d, 1H, J = 8.3 Hz), 4.50 (s,
2H), 4.20 (m, 1H), 3.69 (m, 2H), 1.38 (s, 9H). MS
m/z 348 (MÀÀ1).
(d, 1H, J = 8.3 Hz), 4.62 (m, 2H), 4.46 (m, 1H), 3.98
(m, 1H), 3.76 (m, 1H), 1.43 (s, 9H). MS m/z 361
(M++Na).
5.4.15. N-tert-Butoxycarbonylamino-O-(2,3-dichloroben-
zyl)-D-serine (3n). The title compound was prepared
from 2 and 2,3-dichlorobenzyl bromide by Method A
(yield = 36%). 1H NMR (DMSO-d6) d 7.60 (m, 1H),
7.37 (m, 2H), 6.98 (d, 1H, J = 8.3 Hz), 4.53 (m, 2H),
4.22 (m, 1H), 3.74 (m, 2H), 1.37 (s, 9H). MS m/z 386
(M++Na).
5.4.23. N-tert-Butoxycarbonylamino-O-(2,3,5-trifluoro-
benzyl)-D-serine (3v). The title compound was prepared
from 2 and 2,3,5-trifluorobenzyl bromide by Method
1
A (yield = 98%). H NMR (DMSO-d6) d 7.52 (m, 1H),
7.18 (m, 1H), 6.83 (d, 1H, J = 8.3 Hz), 4.61 (s, 2H),
4.17 (m, 1H), 3.71 (m, 2H), 1.37 (s, 9H). MS m/z 372
(M++Na).
5.4.16. N-tert-Butoxycarbonylamino-O-(2,3-difluorobenzyl)-
D-serine (3o). The title compound was prepared from 2
and 2,3-difluorobenzyl bromide by Method
A
1
(yield = 44%). H NMR (CDCl3) d 7.22–7.00 (m, 3H),
5.39 (d, 1H, J = 7.3 Hz), 4.78 and 4.62 (2m, 2H), 4.48
(m, 1H), 3.97 (dd, 1H, J1 = 2.0 Hz, J2 = 9.3 Hz), 3.77
(dd, 1H, J1 = 3.4 Hz, J2 = 9.3 Hz), 1.45 (s, 9H). MS m/z
330 (MÀÀ1).
5.4.24. N-tert-Butoxycarbonylamino-O-(2,3,6-trifluoro-
benzyl)-D-serine (3w). The title compound was prepared
from 2 and 2,3,6-trifluorobenzyl bromide by Method A
(yield = 84%). 1H NMR (DMSO-d6) d 7.53 (m, 1H),
7.18 (m, 1H), 6.85 (d, 1H, J = 8.3 Hz), 4.63 (s, 2H),
4.17 (m, 1H), 3.71(m, 2H), 1.37 (s, 9H). MS m/z 372
(M++Na).
5.4.17. N-tert-Butoxycarbonylamino-O-(2,4-difluoroben-
zyl)-D-serine (3p). The title compound was prepared
from 2 and 2,4-difluorobenzyl bromide by Method A
5.4.25.
N-tert-Butoxycarbonylamino-O-(2-chloro-3,6-
1
(yield = 86%). H NMR (CDCl3) d 7.32 (m, 1H), 6.90–
difluorobenzyl)-D-serine (3x). The title compound was
prepared from 2 and 2-chloro-3,6-difluorobenzyl bro-
mide by Method A (yield = 69%). H NMR (DMSO-
6.73 (m, 2H), 5.38 (d, 1H, J = 7.9 Hz), 4.56 (s, 2H),
4.47 (m, 1H), 3.95 (dd, 1H, J1 = 2.4 Hz J2 = 9.3 Hz),
3.74 (dd, 1H, J1 = 3.6 Hz J2 = 9.3 Hz), 1.45 (s, 9H).
MS m/z 330 (MÀÀ1).
1
d6) d 7.56 (m, 1H), 7.24 (m, 1H), 6.85 (d, 1H,
J = 8.3 Hz), 4.61 (s, 2H), 4.15 (m, 1H), 3.69 (m, 2H),
1.37 (s, 9H). MS m/z 388 (M++Na).
5.4.18. N-tert-Butoxycarbonylamino-O-(2,5-difluoroben-
zyl)-D-serine (3q). The title compound was prepared
from 2 and 2,5-difluorobenzyl bromide by Method A
(yield = 58%). 1H NMR (CDCl3) d 9.12 (br s, 1H),
7.12–6.87 (m, 3H), 5.44 (d, 1H, J = 8.3 Hz), 4.57 (s,
2H), 4.51 (m, 1H), 3.98 (m, 1H), 3.78 (dd, 1H,
J1 = 3.4 Hz, J2 = 9.3 Hz), 1.45 (s, 9H). MS m/z 330
(MÀÀ1).
5.4.26. N-tert-Butoxycarbonylamino-O-(2-chloro-6-fluo-
robenzyl)-D-serine (3y). The title compound was pre-
pared from 2 and 2-chloro-6-fluorobenzyl bromide by
1
Method A (yield = 41%). H NMR (CDCl3) d 7.50 (br
s, 1H), 7.28–7.16 (m, 2H), 6.99 (m, 1H), 5.40 (d, 1H,
J = 8.1 Hz), 4.70 (s, 2H), 4.45 (m, 1H), 3.96 (m, 1H),
3.74 (m, 1H), 1.44 (s, 9H). MS m/z 346 (MÀÀ1).

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B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
5.4.27.
N-tert-Butoxycarbonylamino-O-(2,6-difluoro-3-
(m, 1H), 4.53 (m, 3H), 4.07 (d, 1H, J = 16.2 Hz), 3.37
(m, 1H), 3.08 (m, 3H), 1.52–1.37 (m, 12H), 0.93 (m,
3H). MS m/z 645 (MH+).
methylbenzyl)-D-serine (3z). The title compound was pre-
pared from 2 and 2,6-difluoro-3-methylbenzyl bromide
by Method A (yield = 98%). H NMR (CDCl3) d 10.2
1
(br s, 1H), 7.09 (m, 1H), 6.77 (m, 1H), 5.38 (d, 1H,
J = 8.3 Hz), 4.62 (s, 2H), 4.44 (m, 1H), 3.94 (m, 1H),
3.73 (dd, 1H, J1 = 3.6 Hz, J2 = 9.5 Hz), 2.23 (s, 3H),
1.43 (s, 9H). MS m/z 368 (M++Na).
5.4.33. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(4-
trifluoromethoxyphenyl)methoxypropionic acid N-(5-(4-
chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothia-
zol-4-ylmethyl)-N-ethylamide hydrochloride (9f). The ti-
tle compound was prepared from 3f as described in
Methods B and C (yield = 16%). 1H NMR (DMSO-
d6) d 7.56 (m, 2H), 7.46–7.32 (m, 6H), 4.65 (m, 1H),
4.52 (d, 1H, J = 16.1 Hz), 4.43 (s, 2H), 4.05 (d, 1H,
J = 16.1 Hz), 3.35 (m, 1H), 3.05 (m, 3H), 1.42 (m,
12H), 0.92 (m, 3H). MS m/z 661 (MH+).
5.4.28. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(4-
fluorophenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9a). The title compound
was prepared from 3a as described in Methods B and C
1
(yield over all steps = 45%). H NMR (CDCl3) d 7.45
(d, 2H, J = 8.4 Hz), 7.38 (d, 2H, J = 8.4 Hz), 7.22 (dd,
2H, J1 = 8.4 Hz, J2 = 5.5 Hz), 7.02 (dd, 2H, J1 = 8.4 Hz,
J2 = 8.7 Hz), 4.89 (dd, 1H, J1 = 6.8 Hz, J2 = 14.0 Hz),
4.41 (s, 2H), 4.35 (m, 2H), 3.46 (m, 2H), 3.20 (m, 2H),
1.57–1.23 (m, 12H), 0.89 (t, 3H). MS m/z 595 (MH+).
5.4.34. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(4-
chloro-2-fluorophenyl)methoxypropionic acid N-(5-(4-
chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothia-
zol-4-ylmethyl)-N-ethylamide hydrochloride (9g). The ti-
tle compound was prepared from 3g as described in
Methods B and C (yield = 25%). 1H NMR (DMSO-
d6) d 7.55 (m, 2H), 7.47–7.27 (m, 5H), 4.65 (m, 1H),
4.48 (m, 3H), 4.06 (d, 1H, J = 16.1 Hz), 3.35 (m, 1H),
3.08 (m, 3H), 1.43 (m, 12H), 0.92 (m, 3H). MS m/z
629 (MH+).
5.4.29. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(3-
fluorophenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9b). The title compound
was prepared from 3b as described in Methods B and C
(yield = 55%). 1H NMR (DMSO-d6) d 7.56 (d, 2H,
J = 8.5 Hz), 7.43 (d, 2H, J = 8.5 Hz), 7.40 (m, 1H), 7.11
(m, 3H), 4.67 (dd, 1H, J1 = 5.1 Hz, J2 = 8.3 Hz), 4.54 (d,
1H, J = 16.2 Hz), 4.43 (s, 2H), 4.04 (d, 1H,
J = 16.2 Hz), 3.36 (dd, 1H, J1 = 8.7 Hz, J2 = 9.7 Hz),
3.08 (m, 3H), 1.46 (s, 3H), 1.44 (s, 3H), 1.41 (s, 3H),
1.40 (s, 3H), 0.93 (t, 3H). MS m/z 595 (MH+).
5.4.35. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2-
chloro-4-fluorophenyl)methoxypropionic acid N-(5-(4-
chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothia-
zol-4-ylmethyl)-N-ethylamide hydrochloride (9h). The ti-
tle compound was prepared from 3h as described in
1
Methods B and C (yield = 28%). H NMR (DMSO-d6)
d 7.56 (m, 2H), 7.43 (m, 4H), 7.23 (m, 1H), 4.64 (m,
1H), 4.55 (d, 1H, J = 16.1 Hz), 4.45 (s, 2H), 4.02 (d,
1H, J = 16.1 Hz), 3.38 (m, 1H), 3.07 (m, 3H), 1.43 (m,
12H), 0.95 (m, 3H). MS m/z 629 (MH+).
5.4.30. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2-
fluorophenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9c). The title compound
was prepared from 3c as described in Methods B and C
(yield = 52%). 1H NMR (DMSO-d6) d 7.56 (m, 2H),
7.42 (m, 2H), 7.38 (m, 2H), 7.20 (m, 2H), 4.64 (m, 1H),
4.51 (m, 3H), 4.03 (d, 1H, J = 16.2 Hz), 3.37 (m, 1H),
3.08 (m, 3H), 1.52–1.36 (m, 12H), 0.93 (m, 3H). MS m/z
595 (MH+).
5.4.36. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(4-
cyanophenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9i). The title compound
was prepared from 3i as described in Methods B and C
1
(yield = 59%). H NMR (DMSO-d6) d 8.27 (br s, 2H),
7.91–7.79 (m, 3H), 7.59–7.52 (m, 2H), 7.49–7.40 (m, 3H),
4.67 (m, 1H), 4.56 (d, 1H, J = 15.9 Hz), 4.51 (s, 2H), 4.06
(d, 1H, J = 15.9 Hz), 3.36 (m, 1H), 3.18–3.01 (m, 3H),
1.53–1.32 (m, 12H), 0.93 (m, 3H). MS m/z 600 (MÀÀH).
5.4.31. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(4-
chlorophenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9d). The title compound
was prepared from 3d as described in Methods B and C
(yield = 38%). 1H NMR (DMSO-d6) d 7.57 (m, 2H),
7.42 (m, 4H), 7.30 (m, 2H), 4.63 (m, 1H), 4.53 (d, 1H,
J = 16.2 Hz), 4.40 (s, 2H), 4.04 (d, 1H, J = 16.2 Hz),
3.35 (m, 1H), 3.06 (m, 3H), 1.53–1.37 (m, 12H), 0.93
(m, 3H). MS m/z 611 (MH+).
5.4.37. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2-
cyanophenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9j). The title compound
was prepared from 3j as described in Methods B and C
(yield = 14%). 1H NMR (DMSO-d6) d 7.87–7.27 (m, 8H),
4.65 (m, 1H), 4.59 (s, 2H), 4.43 (d, 1H, J = 16.1 Hz), 4.15
(d, 1H, J = 16.1 Hz), 3.40 (m, 2H), 3.09 (m, 2H), 1.43–
1.10 (m, 12H), 0.85 (m, 3H). MS m/z 602 (MH+).
5.4.32. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(4-
trifluoromethylphenyl)methoxypropionic acid N-(5-(4-
chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothia-
zol-4-ylmethyl)-N-ethylamide hydrochloride (9e). The ti-
tle compound was prepared from 3e as described in
5.4.38. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2-
trifluoromethylphenyl)methoxypropionic acid N-(5-(4-
chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothia-
zol-4-ylmethyl)-N-ethylamide hydrochloride (9k). The ti-
tle compound was prepared from 3k as described in
1
Methods B and C (yield = 32%). H NMR (DMSO-d6)
d 7.72 (m, 2H), 7.56 (m, 2H), 7.52–7.41 (m, 4H), 4.68

B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
6757
1
Methods B and C (yield = 58%). H NMR (DMSO-d6)
was prepared from 3q as described in Methods B and C
(yield = 40%). 1H NMR (DMSO-d6) d 7.56 (m, 2H),
7.43 (m, 2H), 7.22 (m, 3H), 4.65 (m, 1H), 4.51 (m, 3H),
4.05 (d, 1H, J = 16.3 Hz), 3.38 (m, 1H), 3.10 (m, 3H),
1.52–1.36 (m, 12H), 0.93 (m, 3H). MS m/z 613 (MH+).
d 7.74–7.27 (m, 8H), 4.65 (m, 1H), 4.59 (s, 2H), 4.45 (d,
1H, J = 6.0 Hz), 4.14 (d, 1H, J = 6.0 Hz), 3.38 (m, 2H),
3.19 (m, 2H), 1.43–1.11 (m, 12H), 0.85 (m, 3H). MS m/z
645 (MH+).
5.4.39. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2-
methylphenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9l). The title compound
was prepared from 3l as described in Methods B and C
(yield = 26%). 1H NMR (DMSO-d6) d 7.57 (m, 2H),
7.44 (m, 2H), 7.18 (m, 4H), 4.66 (m, 1H), 4.53 (d, 1H,
J = 16.2 Hz), 4.39 (s, 2H), 4.05 (d, 1H, J = 16.2 Hz),
3.33 (m, 1H), 3.04 (m, 3H), 2.24 (s, 3H), 1.43 (m, 6H),
1.39 (s, 6H), 0.94 (m, 3H). MS m/z 591 (MH+).
5.4.45. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(3,5-
difluorophenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9r). The title compound
was prepared from 3r as described in Methods B and C
(yield = 36%). 1H NMR (DMSO-d6) d 7.56 (m, 2H), 7.43
(m, 2H), 7.13 (m, 1H), 6.68 (m, 2H), 4.66 (m, 1H), 4.48
(m, 3H), 4.04 (d, 1H, J = 16.2 Hz), 3.36 (m, 1H), 3.10 (m,
3H), 1.52–1.36 (m, 12H), 0.93 (m, 3H). MS m/z 613 (MH+).
5.4.46. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2,6-
difluorophenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9s). The title compound
was prepared from 3s as described in Methods B and C
(yield = 27%). 1H NMR (DMSO-d6) d 7.58–7.40 (m, 5H),
7.11 (m, 2H), 4.59 (m, 1H), 4.48 (m, 3H), 4.04 (d, 1H,
J = 16.1 Hz), 3.37 (m, 1H), 3.17 (m, 1H), 3.04 (m, 2H),
1.50–1.32 (m, 12H), 0.89 (m, 3H). MS m/z 613 (MH+).
5.4.40. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2-
carboxamidophenyl)methoxypropionic acid N-(5-(4-chloro-
phenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-
ylmethyl)-N-ethylamide hydrochloride (9m). The title
compound was prepared from 3m as described in
1
Methods B and C (yield = 14%). H NMR (DMSO-
d6) d 7.58–7.29 (m, 8H), 4.69–4.52 (m, 4H), 4.01 (d,
1H, J = 16.1 Hz), 3.35 (m, 1H), 3.10 (m, 3H), 1.45
(m, 6H), 1.40 (m, 6H), 0.94 (m, 3H). MS m/z 620
(MH+).
5.4.47. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2,6-
dichlorophenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9t). The title compound
was prepared from 3t as described in Methods B and C
(yield = 38%). 1H NMR (DMSO-d6) d 7.58–7.24 (m,
7H), 4.62 (m, 3H), 4.41 (d, 1H, J = 16.0 Hz), 4.14 (d,
1H, J = 16.0 Hz), 3.41 (m, 2H), 3.06 (m, 2H), 1.38–1.10
(m, 12H), 0.82 (m, 3H). MS m/z 654 (MH+).
5.4.41. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2,3-
dichlorophenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9n). The title compound
was prepared from 3n as described in Methods B and C
(yield = 34%). 1H NMR (DMSO-d6) d 7.64–7.53 (m, 3H),
7.46–7.30 (m, 4H), 4.68 (m, 1H), 4.58 (m, 1H), 4.52 (s,
2H), 4.04 (d, 1H, J = 16.3 Hz), 3.40 (m, 1H), 3.09 (m,
3H), 1.43 (m, 12H), 0.95 (m, 3H). MS m/z 645 (MH+).
5.4.48. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2,4,
5-trifluorophenyl)methoxypropionic acid N-(5-(4-chlor-
ophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-
ylmethyl)-N-ethylamide hydrochloride (9u). The title com-
pound was prepared from 3u as described in Methods B
5.4.42. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2,3-
difluorophenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9o). The title compound
was prepared from 3o as described in Methods B and C
(yield = 33%). 1H NMR (DMSO-d6) d 7.56 (m, 2H),
7.43 (m, 2H), 7.38 (m, 1H), 7.21 (m, 2H), 4.64 (m, 1H),
4.52 (m, 3H), 4.05 (d, 1H, J = 16.3 Hz), 3.38 (m, 1H),
3.10 (m, 3H), 1.52–1.38 (m, 12H), 0.93 (m, 3H). MS m/z
613 (MH+).
1
and C (yield = 19%). H NMR (DMSO-d6) d 7.65–7.30
(m, 6H), 4.66 (m, 1H), 4.53 (d, 1H, J = 16.2 Hz), 4.42
(m, 2H), 4.07 (d, 1H, J = 16.2 Hz), 3.35 (m, 1H), 3.09
(m, 3H), 1.42 (m, 12H), 0.93 (m, 3H). MS m/z 631 (MH+).
5.4.49. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2,3,
5-trifluorophenyl)methoxypropionic acid N-(5-(4-chlor-
ophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-
ylmethyl)-N-ethylamide hydrochloride (9v). The title com-
pound was prepared from 3v as described in Methods B
5.4.43. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2,4-
difluorophenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9p). The title compound
was prepared from 3p as described in Methods B and C
1
and C (yield = 18%). H NMR (DMSO-d6) d 7.60–7.30
(m, 5H), 7.18 (m, 1H), 4.66 (m, 1H), 4.60–4.45 (m, 3H),
4.06 (d, 1H, J = 16.1 Hz), 3.38 (m, 1H), 3.12 (m, 3H),
1.42 (m, 12H), 0.93 (m, 3H). MS m/z 631 (MH+).
1
(yield = 50%). H NMR (CDCl3) d 7.48–7.22 (m, 5H),
6.82 (m, 2H), 4.89 (m, 2H), 4.47 (m, 2H), 4.28 (m, 1H),
3.53 (m, 2H), 3.22 (m, 2H), 1.61–1.38 (m, 12H), 0.87
(m, 3H). MS m/z 613 (MH+).
5.4.50. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2,3,
6-trifluorophenyl)methoxypropionic acid N-(5-(4-chlor-
ophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-
ylmethyl)-N-ethylamide hydrochloride (9w). The title com-
pound was prepared from 3w as described in Methods B
5.4.44. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2,5-
difluorophenyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (9q). The title compound
1
and C (yield = 32%). H NMR (DMSO-d6) d 7.56 (m,
3H), 7.44 (m, 2H), 7.18 (m, 1H), 4.61 (m, 1H), 4.50 (m,

6758
B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
3H), 4.07 (d, 1H, J = 16.1 Hz), 3.38 (m, 1H), 3.17
(m, 1H), 3.04 (m, 2H), 1.42 (m, 12H), 0.89 (m, 3H). MS
m/z 631 (MH+).
(m, 3H), 4.02 (m, 1H), 3.88 (m, 1H), 1.44 (s, 9H). MS
m/z 295 (MÀÀ1).
5.4.57. N-tert-Butoxycarbonylamino-O-(3,5-dimethyl-iso-
xazol-4-ylmethyl)-D-serine (10d). The title compound was
prepared from 2 and 4-bromomethyl-3,5-dimethyl-isox-
5.4.51. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2-
chloro-3,6-difluorophenyl)methoxypropionic acid N-(5-
(4-chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothia-
zol-4-ylmethyl)-N-ethylamide hydrochloride (9x). The title
compound was prepared from 3x as described in Methods
B and C (yield = 32%). 1H NMR (DMSO-d6) d 7.60–7.29
(m, 6H), 4.61 (m, 1H), 4.44 (s, 2H), 4.48 (d, 1H,
J = 16.2 Hz ), 4.07 (d, 1H, J = 16.2 Hz), 3.38 (m, 1H),
3.17 (m, 1H), 3.05 (m, 2H), 1.49–1.35 (m, 12H), 0.90
(m, 3H). MS m/z 647 (MH+).
1
azole by Method A (yield = 70%). H NMR (CDCl3) d
8.04 (br s, 1H), 5.38 (d, 1H, J = 8.6 Hz), 4.47 (m, 1H),
4.30 (m, 2H), 3.85 (m, 1H), 3.68 (m, 1H), 2.37 (s, 3H),
2.21 (s, 3H), 1.44 (s, 9H). MS m/z 313 (MÀÀ1).
5.4.58. N-tert-Butoxycarbonylamino-O-(2-methyl-thiazol-
4-ylmethyl)-D-serine (10e). The title compound was pre-
pared from 2 and 4-bromomethyl-2-methyl-thiazole by
1
Method A (yield = 51%). H NMR (CDCl3) d 9.30 (br s,
5.4.52. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2-
chloro-6-fluorophenyl)methoxypropionic acid N-(5-(4-
chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-
4-ylmethyl)-N-ethylamide hydrochloride (9y). The title
compound was prepared from 3y as described in Methods
1H), 7.05 (m, 1H), 5.68 (d, 1H, J = 8.1 Hz), 4.63 (s,
2H), 4.47 (m, 1H), 4.02 (m, 1H), 3.78 (dd, 1H,
J1 = 3.6 Hz, J2 = 9.6 Hz), 2.71 (s, 3H), 1.45 (s, 9H). MS
m/z 315 (MÀÀ1).
1
B and C (yield = 50%). H NMR (DMSO-d6) d 7.58 (d,
5.4.59. N-tert-Butoxycarbonylamino-O-thiazol-4-ylmeth-
yl-D-serine (10f). The title compound was prepared from 2
and 4-bromomethyl-thiazole by Method A (yield = 41%).
1H NMR (CDCl3) d 8.88 (br s, 2H), 7.31 (s, 1H), 5.65 (d,
1H, J = 8.1 Hz), 4.73 (m, 2H), 4.48 (m, 1H), 4.03 (m, 1H),
3.80 (dd, 1H, J1 = 3.6 Hz, J2 = 9.7 Hz), 1.44 (s, 9H). MS
m/z 301 (MÀÀ1).
2H, J = 8.5 Hz), 7.50–7.20 (m, 5H), 4.61 (dd, 1H,
J1 = 5.4 Hz, J2 = 8.0 Hz), 4.53 (s, 2H), 4.50 (d, 1H,
J = 16.1 Hz), 4.05 (d, 1H, J = 16.2 Hz), 3.40 (dd, 1H,
J1 = 9.4 Hz, J2 = 8.9 Hz), 3.14 (dd, 1H, J1 = 4.9 Hz,
J2 = 9.9 Hz), 3.05 (m, 2H), 1.42 (m, 6H), 1.39 (s, 3H),
1.38 (s, 3H), 0.91 (t, 3H). MS m/z 629 (MH+).
5.4.53. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(2,6-
difluoro-3-methylphenyl)methoxypropionic acid N-(5-(4-
chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-
4-ylmethyl)-N-ethylamide hydrochloride (9z). The title
compound was prepared from 3z as described in Methods
5.4.60. N-tert-Butoxycarbonylamino-O-thien-3-ylmethyl-
D-serine (10g). The title compound was prepared from 2
and 3-bromomethyl-thiophene by Method A (yield =
30%). 1H NMR (CDCl3) d 8.83 (br s, 1H), 7.33–7.00 (m,
3H), 5.40 (d, 1H, J = 8.1 Hz), 4.72–4.53 (m, 2H), 4.47 (m,
1H), 3.91 (m, 1H), 3.70 (dd, 1H, J1 = 3.3 Hz, J2 =
9.3 Hz), 1.44 (s, 9H). MS m/z 300 (MÀÀ1).
1
B and C (yield = 37%). H NMR (DMSO-d6) d 7.54 (m,
2H), 7.41 (m, 2H), 7.31 (m, 1H), 6.99 (m, 1H), 4.59–
4.45 (m, 4H), 4.04 (d, 1H, J = 16.3 Hz), 3.37 (m, 1H),
3.13 (m, 1H), 3.04 (m, 2H), 2.20 (s, 3H), 1.42 (m, 12H),
0.90 (m, 3H). MS m/z 627 (MH+).
5.4.61. N-tert-Butoxycarbonylamino-O-thien-2-ylmethyl-
D-serine (10h). The title compound was prepared from 2
and 2-bromomethyl-thiophene by Method A (yield =
45%). 1H NMR (CDCl3) d 8.85 (br s, 1H), 7.28 (m, 1H),
6.95 (m, 2H), 5.40 (d, 1H, J = 8.1 Hz), 4.73 (m, 2H), 4.47
(m, 1H), 3.91 (m, 1H), 3.70 (dd, 1H, J1 = 3.3 Hz,
J2 = 9.3 Hz), 1.44 (s, 9H). MS m/z 300 (MÀÀ1).
5.4.54. N-tert-Butoxycarbonylamino-O-pyridin-2-ylmeth-
yl-D-serine (10a). The title compound was prepared from
2 and 2-bromomethyl-pyridine by Method A (yield =
1
62%). H NMR (CDCl3) d 8.94 (br s, 2H), 8.60 (d, 1H,
J = 4.6 Hz), 7.78 (m, 1H), 7.42 (d, 1H, J = 7.9 Hz), 7.30
(m, 1H), 5.72 (d, 1H, J = 7.6 Hz), 4.76 (m, 2H), 4.46 (m,
1H), 4.03 (dd, 1H, J1 = 3.4 Hz, J2 = 10.0 Hz), 3.88 (dd,
1H, J1 = 4.6 Hz, J2 = 10.0 Hz), 1.44 (s, 9H). MS m/z 295
(MÀÀ1).
5.4.62. N-tert-Butoxycarbonylamino-O-(1-phenylethyl)-D-
serine (10i). The title compound was prepared from 2 and
(1-bromoethyl)-benzene by Method A (yield = 50%). H
1
NMR (DMSO-d6) d 12.60 (br s, 1H), 7.30 (m, 5H),
6.85 (d, 1H, J = 8.3 Hz), 4.45 (m, 1H), 3.37 (m, 1H),
3.63 (m, 2H), 1.38 (s, 9H), 1.32 (d, 3H, J = 6.4 Hz). MS
m/z 308 (MÀÀ1).
5.4.55. N-tert-Butoxycarbonylamino-O-pyridin-3-ylmeth-
yl-D-serine (10b). The title compound was prepared from
2 and 3-bromomethyl-pyridine by Method A (yield =
67%). 1H NMR (CDCl3) d 8.60 (br s, 1H), 8.42 (m, 2H),
7.90–7.70 (m, 1H), 7.40 (m, 1H), 7.30 (m, 1H), 5.61 (d,
1H, J = 7.5 Hz), 4.77–4.28 (m, 3H), 4.01 (dd, 1H,
J1 = 2.4 Hz, J2 = 9.2 Hz), 3.82 (m, 1H), 1.44 (s, 9H). MS
m/z 295 (MÀÀ1).
5.4.63. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(pyri-
din-2-yl)methoxypropionic acid N-(5-(4-chlorophenyl)-3,3-
dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-
ethylamide hydrochloride (11a). The title compound was
prepared from 10a as described in Methods B and C
(yield over all steps = 30%). 1H NMR (DMSO-
d6 + D2O) d 8.73 (m, 1H), 8.35 (m, 1H), 7.79 (m, 2H),
7.56 (m, 2H), 7.43 (m, 2H), 4.72 (m, 3H), 4.44 (d, 1H,
J = 16.1 Hz), 4.05 (d, 1H, J = 16.1 Hz), 3.48 (m, 1H),
3.27 (m, 1H), 3.09 (m, 2H), 1.53–1.37 (m, 12H), 0.95
(m, 3H). MS m/z 578 (MH+).
5.4.56. N-tert-Butoxycarbonylamino-O-pyridin-4-ylmeth-
yl-D-serine (10c). The title compound was prepared from
2 and 4-bromomethyl-pyridine by Method A (yield =
1
34%). H NMR (CDCl3) d 9.57 (br s, 2H), 8.49 (m,
2H), 7.35 (m, 2H), 5.60 (d, 1H, J = 7.5 Hz), 4.80–4.42

B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
6759
5.4.64. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(pyri-
din-3-yl)methoxypropionic acid N-(5-(4-chlorophenyl)-3,3-
dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-eth-
ylamide hydrochloride (11b). The title compound was pre-
pared from 10b as described in Methods B and C
prepared from 10g as described in Methods B and C
1
(yield = 29%). H NMR (DMSO-d6 + D2O) d 7.57 (d,
2H, J = 8.5 Hz), 7.51 (dd, 1H, J1 = 3.0 Hz, J2 = 4.9 Hz),
7.43 (d, 2H, J = 8.5 Hz), 7.36 (m, 1H), 7.02 (dd, 1H,
J1 = 1.0 Hz, J2 = 4.9 Hz), 4.62 (m, 1H), 4.55 (d, 1H,
J = 16.2 Hz), 4.40 (s, 2H), 4.02 (d, 1H, J = 16.2 Hz), 3.32
(dd, 1H, dd, 1H, J1 = 8.9 Hz, J2 = 9.9 Hz), 3.03 (m, 3H),
1.45 (s, 3H), 1.43 (s, 3H), 1.40 (m, 6H), 0.92 (t, 3H,
J = 6.8 Hz). MS m/z 583 (MH+).
1
(yield = 30%). H NMR (DMSO-d6 + D2O) d 8.82 (m,
1H), 8.74 (m, 1H), 8.36 (m, 1H), 7.78 (dd, 1H,
J1 = 5.8 Hz, J2 = 7.9 Hz,), 7.58 (m, 2H), 7.43 (m, 2H),
4.66 (m, 3H), 4.52 (d, 1H, J = 16.2 Hz), 4.06 (d, 1H,
J = 16.2 Hz), 3.43 (m, 1H), 3.23 (m, 1H), 3.08 (m, 2H),
1.53–1.37 (m, 12H), 0.93 (m, 3H). MS m/z 578 (MH+).
5.4.70. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(thien-
2-yl)methoxypropionic acid N-(5-(4-chlorophenyl)-3,3-di-
methyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-eth-
ylamide hydrochloride (11h). The title compound was
prepared from 10h as described in Methods B and C (yield
5.4.65. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(pyri-
din-4-yl)methoxypropionic acid N-(5-(4-chlorophenyl)-3,3-
dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-eth-
ylamide hydrochloride (11c). The title compound was pre-
pared from 10c as described in Methods B and C
1
over all steps = 6%). H NMR (CDCl3) d 7.44 (m, 2H),
7.39 (m, 2H), 7.28 (m, 1H), 6.95 (m, 2H), 4.88 (m, 1H),
4.61 (m, 3H), 4.16 (d, 1H, J = 16.2 Hz), 3.49 (m, 2H),
3.28 (m, 1H), 3.14 (m, 1H), 1.52 (s, 3H), 1.47 (s, 3H),
1.31 (s, 3H), 1.28 (s, 3H), 0.89 (m, 3H). MS m/z 583(MH+).
1
(yield = 54%). H NMR (DMSO-d6 + D2O) d 8.85 (m,
2H), 7.87 (m, 2H), 7.57 (m, 2H), 7.44 (m, 2H), 4.75 (m,
3H), 4.53 (d, 1H, J = 16.2 Hz), 4.07 (d, 1H,
J = 16.2 Hz), 3.48 (m, 1H), 3.26 (m, 1H), 3.10 (m, 2H),
1.53–1.38 (m, 12H), 0.95 (m, 3H). MS m/z 578 (MH+).
5.4.71. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(1-
phenylethyl)methoxypropionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (11i and 11j). The racemic
compound 10i was coupled with 410 as described in Method
B, but running the Boc-deprotection with TFA for only 1 h.
The product (68 mg, 0.13 mmol) was then added to a solu-
tion of N-FMOC-a-amino-isobutyric acid (44 mg,
0.13 mmol), TBTU (52 mg, 0.16 mmol), and NEt3 (23 ll,
0.16 mmol) in CH2Cl2 (10 ml) and the mixture was stirred
overnight at rt. The solution was extracted with water and
the organic layer was separated, dried over Na2SO4 and
concentrated. The residue was dissolved in DMF (2 ml)
and ethylamine (1 ml), left for 2 h at rt and was evaporated.
PurificationbyRP-HPLC (YMC Combi PrepODSA 5 lm
120A 20· 50 mm, CH3CN–0.1% TFA in water) yielded the
title compound as a mixture of isomers which was separated
by HPLC (Chiralpak AD, hexane–0.05%TFA/isopropyl
acetate). Yield over all steps = 5%. The NMR spectrum
for both isomers is identical. 1H NMR (CDCl3) d 7.95 (m,
1H), 7.40–7.12 (m, 9H), 4.79 (m, 1H), 4.50 (m, 2H), 4.29
(m, 1H), 4.15 (m, 1H), 3.26 (m, 2H), 3.14 (m, 2H), 1.46
(m, 6H), 1.29 (m, 3H), 1.22 (m, 6H), 0.84 (m, 3H). MS m/
z 591 (MH+).
5.4.66. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(3,5-
dimethyl-isoxazol-4-yl)methoxypropionic acid N-(5-(4-
chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothia-
zol-4-ylmethyl)-N-ethylamide hydrochloride (11d). The
title compound was prepared from 10d as described in
Methods B and C (yield = 54%). 1H NMR (DMSO-
d6 + D2O) d 7.60 (d, 2 H, J = 8.5 Hz), 7.44 (d, 2H,
J = 8.5 Hz), 4.60 (m, 1H), 4.48 (d, 1H, J = 16.2 Hz),
4.21 (s, 2H), 4.05 (d, 1H, J = 16.2 Hz), 3.48 (dd, 1H,
J1 = 8.8 Hz, J2 = 9.5 Hz), 3.07 (m, 3H), 2.33 (s, 3H),
2.14 (s, 3H), 1.50–1.38 (m, 12H), 0.92 (t, 3H,
J = 6.9 Hz). MS m/z 596 (MH+).
5.4.67. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3- (2-
methyl-thiazol-4-yl)methoxypropionic acid N-(5-(4-chlor-
ophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-
ylmethyl)-N-ethylamide hydrochloride (11e). The title
compound was prepared from 10e as described in Meth-
ods B and C (yield = 57%). 1H NMR (DMSO-
d6 + D2O) d 7.56 (m, 2H), 7.43 (m, 2H), 7.34 (s, 1H),
4.64 (m, 1H), 4.54 (d, 1H, J = 16.2 Hz), 4.44 (s, 2H),
4.03 (d, 1H, J = 16.2 Hz), 3.38 (m, 1H), 3.20–3.09 (m,
3H), 2.65 (s, 3H), 1.44 (m, 6H), 1.40 (m, 6H), 0.93 (m,
3H). MS m/z 598 (MH+).
5.4.72. (D)-N-Acetyl-2-amino-5-phenyl-pentanoic acid (14a).
A solution of sodium ethoxide was generated by the addi-
tion of sodium metal (52.9 g, 2.30 mol) over 3 h to EtOH
(1500 ml). To the sodium ethoxide solution at rt was added
a solution of diethylacetamidomalonate (12, 499.75 g,
2.30 mol) dissolved in EtOH (225 ml). The reaction mix-
ture was stirred for 1.5 h at rt, 1-bromo-3-phenylpropane
(458 g, 2.30 mol) was added over 15 min and the mixture
was refluxed for 16 h. The mixture was concentrated to
dryness and the residue partitioned between ethyl acetate
(500 ml) and water (1500 ml). The organic layers were
combined, washed with satd NaCl solution, dried over
Na2SO4 and evaporated to yield 2-acetylamino-2-(3-phe-
nylpropyl)-malonic acid diethyl ester (13a) (752 g, 98%).
5.4.68. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(thia-
zol-4-yl)methoxypropionic acid N-(5-(4-chlorophenyl)-3,3-
dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-eth-
ylamide hydrochloride (11f). The title compound was pre-
pared from 10f as described in Methods B and C
(yield = 52%). 1H NMR (DMSO-d6 + D2O) d 9.74 (d, 1H,
J = 2.0 Hz), 7.57 (m, 3H), 7.43 (d, 2H, J = 8.5 Hz), 4.70–
4.48 (m, 4H), 4.04 (d, 1H, J = 16.2 Hz), 3.40 (m, 1H), 3.19
(m, 1H), 3.05 (m, 2H), 1.53–1.35 (4s, 12H), 0.93 (t, 3H,
J = 6.9 Hz). MS m/z 584 (MH+).
5.4.69. 2-(R)-2-(2-Amino-2-methylpropionylamino)-3-(thien-
3-yl)methoxypropionic acid N-(5-(4-chlorophenyl)-3,3-di-
methyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-eth-
ylamide hydrochloride (11g). The title compound was
A slurry of 13a (249 g, 0.74 mol) and 2.5 M NaOH was
heated at 100 ꢁC for 3 h. The mixture was cooled to

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B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
30 ꢁC and the pH adjusted to 5.0 using concd HCl. The
solution was heated to 100 ꢁC and the pH was held at
5.0 using concd HCl until the reaction was complete.
The hot solution was filtered through diatomaceous
earth. The filtrate was cooled to 5–10 ꢁC and the pH
adjusted to 1.0 using concd HCl. The resulting slurry
was stirred for 1 h at 5 ꢁC, filtered and dried in vacuum
to give 160 g (92%) of (DL)-N-acetyl-2-amino-5-phenyl-
pentanoic acid. A solution of this racemic mixture
(160 g, 0.68 mol), CoCl2 (0.40 g), 2 M KCl solution
(340 ml, 0.68 mol) and water (2900 ml) was adjusted
to pH 8.0 using 2 M KOH. Acylase I (Aspergillus mel-
leus, 14.40 g) was added and the mixture was stirred
for 24 h at 40 ꢁC, maintaining a pH of 8.0 by addition
of 2 M KOH solution. The resulting slurry was filtered,
and the filtrate adjusted to pH 2.0 giving a thick slurry.
The product was isolated by filtration, washed with
hexane (730 ml) and dried in vacuum at 50 ꢁC to yield
prepared from 1-bromo-3-(3,5-difluorophenyl)propane
as described for 14a and 15a (total yield = 28%). 1H
NMR (CDCl3) d 8.90 (br s, 1H), 6.65–6.54 (m, 3H),
5.01 (d, 1H, J = 7.5 Hz), 4.36 (m, 1H), 2.63 (m, 2H),
1.98–1.60 (m, 4H), 1.44 (s, 9H). MS m/z 352
(M++Na).
5.4.76. 2-(tert-Butoxycarbonylamino)-5-(2,6-difluorophe-
nyl)-D-pentanoic acid (15d). The title compound was pre-
pared from 1-bromo-3-(2,6-difluorophenyl)propane as
described for 14a and 15a (total yield = 17%). 1H
NMR (CDCl3) d 8.93 (br s, 1H), 7.29 (m, 1H), 7.10–6-
99 (m, 2H), 5.05 (d, 1H, J = 7.5 Hz), 4.38 (m, 1H),
2.65 (m, 2H), 2-08–1.67 (m, 4H), 1.42 (s, 9H). MS m/z
352 (M++Na).
5.4.77. 2-(R)-2-(2-Amino-2-methylpropionylamino)-5-phe-
nylpentanoic acid N-(5-(4-chlorophenyl)-3,3-dimethyl-1,1-
dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-ethylamide hydro-
chloride (16a). The title compound was prepared from
1
14a (68.9 g, 43%). H NMR (DMSO-d6) d 12.39 (br s,
1H), 8.02 (d, 1H), 7.30–7.16 (m, 5H), 4.24 (m, 1H),
2.59 (m, 2H), 1.86 (s, 3H), 1.74–1.59 (m, 4H). MS m/
z 236 (MH+).
1
15a as described in Methods B and C (yield = 91%). H
NMR (DMSO-d6) d 8.39 (d, 1H, J = 8.0 Hz), 8.18 (br s,
3H), 7.86 (s, 1H), 7.52 (d, 2H, J = 8.6 Hz), 7.40 (d, 2H,
J = 8.6 Hz), 7.29 (m, 2H), 7.22–7.12 (m, 3H), 4.64 (d,
1H, J = 16.3 Hz), 4.35 (ddd, 1H), 3.92 (d, 1H,
J = 16.4 Hz), 2.91 (m, 2H), 2.48 (m, 2H), 1.50 (m, 2H),
1.46, 1.43, 1.41, 1.39 (4s, 12H), 1.28 (m, 2H), 0.95 (t,
3H, J = 6.9 Hz). MS m/z 576 (M++H).
5.4.73. 2-(tert-Butoxycarbonylamino)-5-phenyl-D-penta-
noic acid (15a). A solution of 14a (188.5 g, 0.80 mol) in
EtOH (535 ml) and concd HCl (318 ml, 3.80 mol) was
warmed to 85 ꢁC for 22 h. Water was azeotropically
distilled from the reaction by continuous addition and
distillation of EtOH (8000 ml). The EtOH was azeotrop-
ically distilled from the reaction by continuous addition
and distillation of ethyl acetate (2000 ml). The solution
was then stirred at 0 ꢁC for 1 h, the crystallised product
was filtered and dried in vacuum at 40 ꢁC to give (D)-2-
amino-5-phenylpentanoic acid, ethyl ester hydrochloride
(199 g, 96%).
5.4.78. 2-(R)-2-(2-Amino-2-methylpropionylamino)-5-(4-
fluorophenyl)-pentanoic acid N-(5-(4-chlorophenyl)-3,3-di-
methyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-eth-
ylamide hydrochloride (16b). The title compound was
prepared from 15b as described in Methods B and C
1
(yield = 15%). H NMR (DMSO-d6 + D2O) d 7.52 (m,
2H), 7.40 (m, 2H), 7.20–7.05 (m, 4H), 4.51 (d, 1H,
J = 16.4 Hz), 4.34 (m, 1H), 4.07 (d, 1H, J = 16.4 Hz),
2.96 (m, 2H), 2.50 (d, 2H), 2.00 (m, 2H), 1.45 (m, 2H),
1.39 (s, 6H), 1.13 (2s, 6H), 0.87 (t, 3H). MS m/z 594
(M++H).
To a slurry of (D)-2-amino-5-phenylpentanoic acid, eth-
yl ester hydrochloride (10 g, 38.3 mmol) in THF
(400 ml) were added triethylamine (10.7 ml, 76 mmol)
and di-tert-butyl-dicarbonate (10.9 g, 50 mmol), the
mixture was stirred for 18 h at rt, evaporated and dis-
solved in CH2Cl2. After extraction with 0.1 M citric
acid, the organic layer was dried (Na2SO4) and concen-
trated. The crude product was suspended in 2.5 M
NaOH and heated to 60 ꢁC for 5 h. The mixture was
cooled to rt, neutralised with concd HCl and extracted
with CH2Cl2. The organic layer was dried (Na2SO4)
and concentrated to yield 15a (7.30 g, 65%). 1H
NMR (DMSO-d6) d 12.4 (br s, 1H), 7.42 (d, 1H,
J = 7.5 Hz), 7.30–7.08 (m, 5H), 4.27 (m, 1H), 2.58
(m, 2H), 1.57–1.75 (m, 4H), 1.35 (s, 9H). MS m/z
316 (M++Na).
5.4.79. 2-(R)-2-(2-Amino-2-methylpropionylamino)-5-(3,5-
difluorophenyl)-pentanoic acid N-(5-(4-chlorophenyl)-3,3-
dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-eth-
ylamide hydrochloride (16c). The title compound was
prepared from 15c as described in Methods B and C
(yield = 43%). 1H NMR (CDCl3) d 8.01 (d, 1H,
J = 9.0 Hz), 7.46–7.35 (m, 4H), 6.70–6.59 (m, 3H), 4.65–
4.52 (m, 2H), 4.20 (d, 1H, J = 15.7 Hz), 3.18 (m, 1H),
2.97 (m, 1H), 2.57 (m, 2H), 1.59 (m, 2H), 1.57 (2s, 6H),
1.45–1.23 (m, 8H), 0.91 (t, 3H). MS m/z 612 (M++H).
5.4.80. 2-(R)-2-(2-Amino-2-methylpropionylamino)-5-(2,6-
difluorophenyl)- pentanoic acid N-(5-(4-chlorophenyl)-3,3-
dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-eth-
ylamide hydrochloride (16d). The title compound was pre-
pared from 15d as described in Methods B and C
5.4.74. 2-(tert-Butoxycarbonylamino)-5-(4-fluorophenyl)-
D-pentanoic acid (15b). The title compound was pre-
pared from 1-bromo-3-(4-fluorophenyl)propane as de-
1
scribed for 14a and 15a (total yield = 25%). H NMR
1
(CDCl3) d 8.87 (br s, 1H), 7.32–7.03 (m, 4H), 4.99 (d,
1H, J = 7.5 Hz), 4.35 (m, 1H), 2.66 (m, 2H), 1.95–1.60
(m, 4H), 1.44 (s, 9H). MS m/z 334 (M++Na).
(yield = 33%). H NMR (DMSO-d6 + D2O) d 7.50 (m,
2H), 7.40 (m, 2H), 7.29 (m, 1H), 7.04 (m, 2H), 4.51 (d,
1H, J = 16.3 Hz), 4.34 (m, 1H), 4.07 (d, 1H, J = 16.3 Hz),
2.99 (m, 2H), 2.55 (m, 2H), 2.05 (m, 2H), 1.44 (m, 2H),
1.39 (2s, 6H), 1.13 (2s, 6H), 0.85 (t, 3H). MS m/z 612
(M++H).
5.4.75. 2-(tert-Butoxycarbonylamino)-5-(3,5-difluorophe-
nyl)-D-pentanoic acid (15c). The title compound was

B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
6761
5.4.81. 2-(R)-3-(R)-2-(2-Amino-2-methylpropionylamino)-
3-benzyloxy-3-methyl- propionic acid N-(5-(4-chlorophe-
nyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmeth-
yl)-N-ethylamide hydrochloride (18). The title compound
was prepared from N-Boc-O-benzyl-D-allo-threonine
a suspension of methyl 2-amino-3-fluoro-2-fluoromethyl-
propionate hydrochloride9 (250 mg, 1.32 mmol) in aceto-
nitrile (10 ml) was added Me4NOHÆ5H2O (400 mg,
2.20 mmol). The mixture was stirred at rt under Ar for
30 min and then di-tert-butyl-dicarbonate (432 mg,
1.98 mmol) was added. The mixture was stirred for
48 h, the solvent was evaporated and the residue was par-
titioned between water and diethyl ether. The aqueous
layer was washed with ether, acidified with solid citric
acid to pH 3–4 and extracted with ethyl acetate. The com-
bined organic layers were dried (Na2SO4) and evaporated
to yield N-Boc-3-fluoro-2-fluoromethyl-alanine (294 mg,
92%) as a white solid.
1
(17) as described in Methods B and C (yield = 18%). H
NMR (DMSO-d6 + D2O) d 7.62 (m, 2H), 7.48 (m, 2H),
7.38–7.24 (m, 5H), 4.63 (m, 1H), 4.53 (m, 2H), 4.41 (m,
1H), 4.05 (d, 1H, J = 16.3 Hz), 3.86 (m, 1H), 3.39 (m,
1H), 3.01 (m, 1H), 1.59–1.28 (m, 12H), 1.07 (d, 3H,
J = 6.0 Hz), 0.75 (m, 3H). MS m/z 591 (MH+).
5.4.82. N-Boc-N-methyl-O-benzyl-D-serine (21). A sus-
pension of N-Boc-O-benzyl-D-serine (19, 1.95 g,
6.59 mmol), p-toluenesulfonic acid (600 mg, 3.16 mmol)
and paraformaldehyde (1.04 g, 34.4 mmol) was stirred
at 100 ꢁC for 50 min. The solution was cooled to rt,
diluted with ethyl acetate and extracted with water
and NaHCO3. The organic layer was dried (Na2SO4)
and concentrated to yield 20 as a crystalline solid
(884 mg, 44%). The product (884 mg, 2.88 mmol) was
dissolved in chloroform (15 ml), triethylsilane (2.5 ml,
15.7 mmol) and trifluoroacetic acid (10 ml) were added
and the solution was stirred at rt for 6 h. The mixture
was concentrated, dissolved in isohexane and extracted
with satd NaHCO3 solution. The pH was then adjusted
to 3.0 using 10% KHSO4, and the aqueous layer, was
extracted with ethyl acetate. After concentration of
the aqueous layer the crude product was suspended
in THF (60 ml), NEt3 (5 ml, 35.6 mmol) and di-tert-bu-
tyl-dicarbonate (825 mg, 3.8 mmol) were added and the
mixture was stirred overnight at rt. The solvent was
evaporated, the residue was dissolved in tert-butyl-
methyl ether and extracted with water. After acidificat-
ion with 10% citric acid, the aqueous layer was extract-
ed with ethyl acetate, the organic layer was dried
(Na2SO4) and concentrated to yield the title compound
as an amorphous solid (483 mg, 54%). 1H NMR
(CDCl3) d 9.66 (br s, 1H), 7.37–7.28 (m, 5H), 4.95
(m, 1H), 4.55 (m, 2H), 3.90 (m, 2H), 2.94 (s, 3H),
1.44 (s, 9H). MS m/z 308 (MÀÀ1).
The title compound was prepared from 6s and N-Boc-3-
fluoro-2-fluoromethyl-alanine as described in Method C
(yield = 56%). 1H NMR (DMSO-d6) d 9.01 (m, 3H),
7.86 (s, 1H), 7.59–7.39 (m, 5H), 7.12 (m, 2H), 5.13–
4.62 (m, 5H), 4.48 (m, 3H), 4.09 (d, 1H, J = 16.0 Hz),
3.29–3.16 (m, 2H), 3.04 (m, 2H), 1.37 (s, 6H), 0.87 (t,
3H). MS m/z 650 (MH+).
5.4.85. 2-(R)-2-((1-Amino-cyclopropanecarbonyl)-amino)-
3-phenylmethoxypropionic acid N-(5-(4-chlorophenyl)-3,3-
dimethyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-eth-
ylamide hydrochloride (25). The title compound was pre-
pared from 6 (X = H) and 1-(Boc-amino)cyclopropane-
carboxylic acid as described in Method C (yield = 38%).
1H NMR (DMSO-d6 + D2O) d 7.54 (m, 2H), 7.42 (m,
2H), 7.38–7.23 (m, 5H), 4.64 (m, 1H), 4.43 (m, 3H),
4.12 (d, 1H, J = 16.2 Hz), 3.34 (m, 2H), 3.04 (m, 2H),
1.39 (m, 6H), 1.04 (m, 2H), 0.85 (m, 2H), 0.79 (m,
3H). MS m/z 575 (MH+).
5.4.86. 2-(R)-2-(2-(R)-2-Amino-2-methyl-3-hydroxypro-
pionylamino)-3- phenylmethoxypropionic acid N-(5-(4-
chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothia-
zol-4-ylmethyl)-N-ethylamide hydrochloride (26). The
title compound was prepared from 6 (X = H) and
N-Boc-a-methyl-D-serine as described in Method C
(yield = 71%). 1H NMR (DMSO-d6) d 8.52 (d, 1H,
J = 7.9 Hz), 8.12 (br s, 3H), 7.87 (br s, 1H), 7.56
(m, 2H), 7.45 (m, 2H), 7.39–7.25 (m, 5H), 5.58 (m,
1H), 4.71 (m, 1H), 4.52 (d, 1H, J = 16.0 Hz), 4.41
(s, 2H), 4.06 (d, 1H, J = 16.0 Hz), 3.72 (m, 1H),
3.62 (m, 1H), 3.35 (m, 1H), 3.13 (m, 1H), 3.05 (m,
2H), 1.47–1.32 (m, 9H), 0.90 (m, 3H). MS m/z 593
(MH+).
5.4.83. 2-(R)-2-[2-(R)-2-Aminopropionyl(N-methylamino)]-
3-benzyloxypropionic acid N-(5-(4-chloro phenyl)-3,3-di-
methyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-eth-
ylamide hydrochloride (22) and 2-(R)-2-[2-(S)-2-Amino-
propionyl(N-methylamino)]-3-benzyloxypropionic acid N-
(5-(4-chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroi-
sothiazol-4-ylmethyl)-N-ethylamide hydrochloride (23).
The title compounds were prepared as described in
Methods B and C using 21 and N-Boc-D-alanine to ob-
tain 22 (yield = 90%) or 21 and N-Boc-L-alanine to ob-
tain 23 (yield = 98%). The NMR spectra for 22 and 23
5.4.87. 2-(R)-2-(2-(S)-2-Amino-2-methyl-3-hydroxypro-
pionylamino)-3- phenylmethoxypropionic acid N-(5-(4-
chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroisothia-
zol-4-ylmethyl)-N-ethylamide hydrochloride (27). The
title compound was prepared from 6 (X = H) and
N-Boc-a-methyl-L-serine as described in Method C
(yield = 71%). 1H NMR (DMSO-d6) d 8.52 (d, 1H,
J = 7.9 Hz), 8.10 (br s, 3H), 7.88 (br s, 1H), 7.56
(m, 2H), 7.45 (m, 2H), 7.39–7.25 (m, 5H), 5.65 (m,
1H), 4.71 (m, 1H), 4.52 (d, 1H, J = 16.0 Hz), 4.42
(s, 2H), 4.07 (d, 1H, J = 16.0 Hz), 3.77 (m, 1H),
3.61 (m, 1H), 3.35 (m, 1H), 3.18 (m, 1H), 3.07 (m,
2H), 1.47–1.32 (m, 9H), 0.90 (m, 3H). MS m/z 593
(MH+).
1
are identical. H NMR (CDCl3) d 7.46-7.23 (m, 9H),
5.43 (m, 1H), 4.61–4.36 (m, 3H), 4.18 (m, 1H), 3.83–
3.62 (m, 2H), 3.45 (m, 1H), 3.27 (m, 1H), 3.07 (m,
1H), 2.93 (s, 3H), 1.52–1.44 (m, 6H), 1.18 (m, 3H),
0.86 (m, 3H). MS m/z 576 (MÀÀ1).
5.4.84. 2-(R)-2-(2-Amino-3-fluoro-2-fluoromethylpropio-
nylamino)-3-(2,6- difluorophenyl)methoxy propionic acid
N-(5-(4-chlorophenyl)-3,3-dimethyl-1,1-dioxo-2,3-dihydroi-
sothiazol-4-ylmethyl)-N-ethylamide hydrochloride (24). To

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B. Evers et al. / Bioorg. Med. Chem. 13 (2005) 6748–6762
5.4.88. 2-(R)-2-(2-(R)-2-Aminopropionylamino)-3-phenyl-
methoxypropionic acid N-(5-(4-chloro-phenyl)-3,3-di-
methyl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-eth-
ylamide (28). The title compound was prepared from 6
and N-Boc-D-alanine as described in Method C, but iso-
lated after extraction of the ether layer with NaHCO3 as
the free base (yield = 43%). 1H NMR (CDCl3) d 7.77 (m,
1H), 7.47–7.19 (m, 8H), 4.96 (m, 1H), 4.64 (m, 1H), 4.46
(s, 2H), 4.33 (m, 1H), 4.15 (m, 1H), 3.55–3.39 (m, 3H),
3.30 (m, 1H), 3.09 (m, 1H), 1.55–1.43 (m, 6H), 1.29
(t, 3H), 0.89 (m, 3H). MS m/z 564 (MH+)
challenged to secrete GH by the addition of GH secret-
agogues to the medium. After 45 min at room tempera-
ture, the medium was removed, filtered and stored
frozen until radioimmunoassays for rat GH were per-
formed. EC50 values were calculated as an average of
at least three runs and are represented as means stan-
dard error, unless otherwise stated. All presented com-
pounds caused a release of GH as a statistically
significant increase (at least 20%) of the basal level.
References and notes
5.4.89. 2-(R)-2-(2-(S)-2-Aminopropionylamino)-3-phenyl-
methoxypropionic acid N-(5-(4-chlorophenyl)-3,3-dimeth-
yl-1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-ethyla-
mide (29). The title compound was prepared from 6 and
1. Bowers, C. Y.; Momany, F. A.; Reynolds, G. A.; Hong,
A. Endocrinology 1984, 114, 1537.
2. DeVita, R. J.; Schoen, W. R.; Fisher, M. H.; Frontier, A.
J.; Pisano, J. M.; Wyvratt, M. J.; Cheng, K.; Chan, W.
W.-S.; Butler, B. S.; Hickey, G. J.; Jacks, T. M.; Smith,
R. G. Bioorg. Med. Chem. Lett. 1994, 4, 2249.
3. Kojima, M.; Hosoda, H.; Date, Y. Nature 1999, 402, 656.
4. Carpino, P. A. Expert Opin. Ther. Patents 2002, 12, 1599.
5. Seyler, D. E.; Dodge, J. A.; Osborne, J. J.; Cox, K. L.;
Viswanath, D.; Wilmot, A. F.; Keaton, M. J.; Heiman, M.
L.; Bryant, H. U.; Cutler, G. B., Jr. Drug Dev. Res. 2000,
49, 260.
6. Lugar, C. W.; Clay, M. P.; Lindstrom, T. D.; Woodson,
A. L.; Smiley, D.; Heiman, M. L.; Dodge, J. A. Bioorg.
Med. Chem. Lett. 2004, 14, 5873.
7. Patchett, A. A.; Nargund, R. P.; Tata, J. R.; Chen, M.-H.;
Barakat, K. J.; Johnston, D. B. R.; Cheng, K.; Chan, W.
W.-S.; Butler, B.; Hickey, G.; Jacks, T.; Schleim, K.; Pong,
S.; Chaung, L.-Y. P.; Chen, H. Y.; Frazier, E.; Leung, K.
H.; Chiu, S.-H. L.; Smith, R. G. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 7001.
8. Pan, L. C.; Carpino, P. A.; Lefker, B. A.; Ragan, J. A.;
Toler, S. M.; Pettersen, J. C.; Nettleton, D. O.; Ng, O.;
Pirie, C. M.; Chidsey-Frink, K.; Lu, B.; Nickerson, D. F.;
Tess, D. A.; Mullins, M. A.; MacLean, D. B.; Da Silva-
Jardine, P. A.; Thompson, D. D. Endocrine 2001, 14, 437.
9. Dodge, J. A.; Heiman, M. L. Annu. Rep. Med. Chem.
2003, 38, 81.
10. Dodge, J. A.; Evers, B.; Jungheim, L. N.; Muehl, B. S.;
Ruehter, G.; Thrasher, K. J. WO 02/032888, 2002.
11. Evers, B.; Ruehter, G.; Tebbe, M. J.; Martin de la Nava,
E. M. WO 03/087069, 2003.
1
N-Boc-L-alanine as described for 28 (yield = 40%). H
NMR (CDCl3) d 7.77 (m, 1H), 7.47–7.19 (m, 8H),
4.96 (m, 1H), 4.64 (m, 1H), 4.46 (s, 2H), 4.33 (m,
1H), 4.15 (m, 1H), 3.55–3.39 (m, 3H), 3.30 (m, 1H),
3.09 (m, 1H), 1.55–1.43 (m, 6H), 1.29 (t, 3H), 0.89
(m, 3H). MS m/z 564 (MH+).
5.4.90. 2-(R)-2-(2-Amino-2-methylpropionylamino)-4-phen-
ylmethoxybutyric acid N-(5-(4-chlorophenyl)-3,3-dimethyl-
1,1-dioxo-2,3-dihydroisothiazol-4-ylmethyl)-N-ethylamide
hydrochloride (30). The title compound was prepared
from N-Boc-O-benzyl-D-homoserine as described in
Methods B and C (yield = 9%). 1H NMR (DMSO-
d6 + D2O) d 7.56 (m, 2H), 7.44 (m, 2H), 7.38–7.25 (m,
5H), 4.61 (m, 2H), 4.40 (m, 2H), 3.94 (d, 1H,
J = 16.3 Hz), 3.33 (m, 2H), 3.03 (m, 2H), 1.58–1.32 (m,
14H), 0.95 (m, 3H). MS m/z 591 (MH+).
5.4.91. 2-(R)-2-(2-Amino-2-methylpropionylamino)-4-phen-
ylbutyric acid N-(5-(4-chlorophenyl)-3,3-dimethyl-1,1-dioxo-
2,3-dihydroisothiazol-4-ylmethyl)-N-ethylamide hydrochlo-
ride (31). The title compound was prepared from N-
Boc-D-homophenylalanine as described in Methods B
and C (yield = 51%). 1H NMR (DMSO-d6 + D2O) d
7.50 (m, 2H), 7.39 (m, 2H), 7.32–7.10 (m, 5H), 4.49 (d,
1H, J = 16.3 Hz), 4.30 (m, 1H), 4.04 (m, 1H), 2.84 (m,
1H), 2.69 (m, 1H), 2.40 (m, 2H), 1.59 (m, 1H), 1.42–
1.34 (m, 6H), 1.23–1.13 (m, 7H), 0.75 (m, 3H). MS
m/z 561 (MH+).
12. Evers, B.; Ruehter, G.; Martin de la Nava, E. M.; Tebbe,
M. J. WO 03/087070, 2003.
13. Chenault, H. K.; Dahmer, J.; Whitesides, G. M. J. Am.
Chem. Soc. 1989, 111, 6354.
14. Freidinger, R. M.; Hinkle, J. S.; Perlow, D. S.; Arison, B.
H. J. Org. Chem. 1983, 48, 77.
5.5. GH release in pituitary cells
15. Davis, F. A.; Reddy, G. V.; Bental, M.; Deutsch, C. J.
Synthesis 1994, 701.
16. Palucki, B. L.; Feighner, S. D.; Pong, S.-S.; McKee, K. K.;
Hreniuk, D. L.; Tan, C.; Howard, A. D.; Van der Ploeg,
L. H. Y.; Patchett, A. A.; Nargund, R. P. Bioorg. Med.
Chem. Lett. 2001, 11, 1955.
17. Chen, M.-H.; Steiner, M. G.; Patchett, A. A.; Cheng, K.;
Wei, L.; Chan, W. W.-S.; Butler, B.; Jacks, T. M.; Smith,
R. G. Bioorg. Med. Chem. Lett. 1996, 6, 2163.
Anterior pituitaries from male Sprague–Dawley rats
were sectioned into small pieces and digested enzymati-
cally using trypsin (Difco). Pituitary cells were dispersed
by mechanical agitation, collected, pooled and then
seeded into 96-well plates (50,000 cells/well). After 5
days of culture, the cells formed as monolayer (70–
80% confluent). Cells were then washed with medium
and incubated for 90 min at 37 ꢁC. Then the cells were