Azetidinones as vasopressin V1a antagonists

Article abstract of DOI:10.1016/j.bmc.2006.12.031

The azetidinone LY307174 (1) was identified as a screening lead for the vasopressin V1a receptor (IC₅₀ 45 nM at the human V1a receptor) based on molecular similarity to ketoconazole (2), a known antagonist of the luteinizing hormone releasing h

Full text of DOI:10.1016/j.bmc.2006.12.031

Bioorganic & Medicinal Chemistry 15 (2007) 2054–2080
Azetidinones as vasopressin V1a antagonists
b
b,
Christophe D. Guillon,a,b, Gary A. Koppel, Michael J. Brownstein,
*
Michael O. Chaney,a Craig F. Ferris,c Shi-fang Lu,b,d Karine M. Fabio,a
Marvin J. Miller,e Ned D. Heindel,a David C. Hunden,f Robin D. G. Cooper,f
Stephen W. Kaldor,f,à Jeffrey J. Skelton,f,§ Bruce A. Dressman,f Michael P. Clay,f
Mitchell I. Steinberg,f Robert F. Brunsf and Neal G. Simonb,d
aDepartment of Chemistry, 6 East Packer Avenue, Lehigh University, Bethlehem, PA 18015, USA
bAzevan Pharmaceuticals, Inc., 115 Research Drive, Bethlehem, PA 18015, USA
cDepartment of Psychiatry, University of Massachusetts Medical School, S 7-831, 55 Lake Avenue North, Worcester, MA 01655, USA
dDepartment of Biological Sciences, 111 Iacocca Hall, Lehigh University, Bethlehem, PA 18015, USA
eDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
fLilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 2 October 2006; revised 15 December 2006; accepted 21 December 2006
Available online 23 December 2006
Abstract—The azetidinone LY307174 (1) was identified as a screening lead for the vasopressin V1a receptor (IC50 45 nM at the
human V1a receptor) based on molecular similarity to ketoconazole (2), a known antagonist of the luteinizing hormone releasing
hormone receptor. Structure–activity relationships for the series were explored to optimize receptor affinity and pharmacokinetic
properties, resulting in compounds with Ki values <1 nM and brain levels after oral dosing ꢀ100-fold higher than receptor affinities.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
The neurohypophysical hormones vasopressin1 and oxy-
tocin exert a wide range of physiological effects through
Keywords: Vasopressin; Vasopressin V1a antagonists; Azetidinone;
Oral bioavailability; CNS penetration.
*
Corresponding author. Tel.: +1 610 758 4706; fax: +1 610 758
6536; e-mail: chg3@lehigh.edu
binding to specific membrane receptors belonging to the
G protein-coupled receptor (GPCR) superfamily. To
date, three vasopressin receptor subtypes and one oxyto-
cin receptor have been pharmacologically and function-
ally described.1 V1a, V1b, and oxytocin receptors
activate phospholipase C, resulting in the production
of inositol 1,4,5-trisphosphate and diacylglycerol,
Present address: J. Craig Venter Institute, 9704 Medical Center Dr.,
Rockville, MD 20850, USA.
à
Present address: Takeda San Diego, Inc., 10410 Science Center
Drive, San Diego, CA 92121, USA.
§
Present address: Cargill, Inc., PO Box 5624, Minneapolis, MN 55440,
USA.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.031

C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
2055
mobilization of intracellular calcium, and activation of
protein kinase C. V2 receptors stimulate adenylyl cyclase,
resulting in the accumulation of cyclic AMP and activa-
tion of protein kinase A. All four receptor subtypes
from several mammalian species have been recently
cloned,2–5 as well as closely related receptors from bony
fishes and invertebrates.6,7 Although vasopressin is per-
haps best-known for its role in the cardiovascular system,
it also has actions in the central nervous system (CNS),
and several CNS applications of vasopressin receptor
antagonists have been suggested (reviewed in Refs. 8
and 9). A number of research groups have prepared
antagonists directed at the vasopressin V1 receptor.10–15
While V1a antagonists have been made, none of these
have been reported to penetrate the CNS efficiently.
mutation in A, resulting in a compound with high affin-
ity for B, that is, beta.16,17
In the construction of the Neuropeptide Cassette, 26
known nonpeptide ligands for neuropeptide receptors
(cholecystokinin, substance P, angiotensin II, opiate,
leuteinizing hormone releasing hormone (LHRH), and
motilin receptors) were used in the role of alpha, and
molecular similarity searches in MACCS against the
26 alphas were used to identify ‘mutated’ versions as
candidates for beta. Similarity thresholds were adjusted
to obtain ꢀ300 matches for each of the 26 query struc-
tures, and the ꢀ5000 unique structures selected by this
method were reduced to 1500 using the leader clustering
method.
Among the 26 query structures was ketoconazole, a
marketed antifungal agent known to cause reproductive
side-effects due to antagonism of the LHRH receptor.18
The azetidinone LY307174 (1, Fig. 2) was selected for
screening based on 59% similarity to ketoconazole (2).
Compound 1 was found to have an affinity of 45 nM
for the cloned human V1a receptor. In agreement with
the rationale in Figure 1, some features of 2 such as
the dioxolane ring and terminal phenyl are conserved
in 1, while others are totally replaced.
2. Results and discussion
Our vasopressin antagonist program was initiated to
identify a CNS-active V1a antagonist: one with potent
affinity for the human V1a receptor (IC50 < 10 nM),
good oral availability, and ability to penetrate the blood
brain barrier—in short, a candidate for human clinical
development targeting CNS disorders. The program be-
gan at Lilly in 1990 with the selection of a 1500-com-
pound ‘Neuropeptide Cassette’—a library intended to
identify nonpeptide ligands for neuropeptide receptors.
The library applied the concept of receptor crosstalk—
previously well documented for the biogenic amine sub-
family of GPCRs—to the neuropeptide receptors. The
underlying concept is illustrated in Figure 1. Suppose
there are two related receptors, A and B. Over the
course of evolution, the two receptors have remained
identical in part of the binding site (shown as the semi-
circular portion), while in the rest of the binding site the
two receptors have drifted apart due to mutations
(shown as the triangular portion in A and the rectangu-
lar portion in B). Furthermore, suppose that alpha, a
high-affinity ligand for A, is already known, and we wish
to find beta, a high-affinity ligand for B. One approach
would be to screen all available analogs of alpha in the
hope that a mutation in alpha would complement the
Beyond the simple issue of affinity, 1 was considered an
attractive lead for several reasons. LY307174 is a mono-
cyclic beta-lactam (monobactam). Unlike fused-ring
beta-lactams such as penicillins and cephalosporins,
simple monobactams such as 1 are highly stable to
chemical or enzymatic hydrolysis of the four-membered
azetidinone ring. The cis geometry of the rigid four-
membered ring forces the three side-chains together into
a fixed geometric configuration, presumably enabling
complementarity with sub-pockets of the receptor.
Although the molecular weight of 1 is relatively high
(568.6), its compactness provided some hope that the
series might show significant oral absorption.
After our lead compound was identified, we undertook a
structure–activity relationship (SAR) study utilizing the
chiral Staudinger 2 + 2 cycloaddition reaction as depict-
ed with a representative example in Scheme 1.
In the sequence in Scheme 1, trans-cinnamaldehyde is
combined with glycine benzyl ester to form the imine
3, which is then reacted with the chiral auxiliary acid
chloride 4, to form the four-membered ring 5. In the
example given in Scheme 1, the S-chiral auxiliary induc-
es formation of the R-cis configuration in the azetidi-
none product. The methylene group of the glycine
constituent could be acylated, producing 6, which dis-
played potent activity (V1a IC50 = 6.5 nM). The abso-
lute stereochemistry of the chiral methine carbons of
the azetidinone ring of 6, depicted in Scheme 1, was as-
signed based on earlier reports on the absolute configu-
ration of these azetidinone carbons produced through
the Staudinger 2 + 2 reaction with the Evans’ chiral aux-
iliary.19,20,25 Confirming this assignment, the analog 7
was crystallized and the absolute configuration deter-
mined by X-ray analysis21 as shown in Figure 3.
Ligand
Receptor
Tight fit
Loose fit
Ligand
Receptor
Loose fit
Tight fit
Figure 1. Receptor crosstalk as a screening tool.

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C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
O
N
O
O
N
N
O
O
N
O
O
O
O
O
N
N
O
ketoconazole (2)
IC50 = 2 μM
LHRH receptor
LY307174 (1)
IC50 = 45 nM
human V1a receptor
Figure 2.
O
O
O
N
+
COCl
H2N
CO2Bz
CH2Cl2 / MgSO4
> 90%
3
4
N
CO2Bz
Et3N / CH2Cl2 / 0OC
60-95%
O
O
O
O
1) LDA / THF / -78OC
N
O
N
O
2) PIVALOYL
CHLORIDE
90%
N
N
O
CO2Bz
CO2Bz
6
5
Scheme 1.
O
O
N
S
S
R
O
R
N
O
O
O
Me3Si
O
Figure 3. X-ray structure/absolute configuration of 7.

C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
2057
O
O
O
A
B
N
O
H
N
O
N
O
O
O
O
N
C
O
N
N
O
O
8
9
IC50 = 61 μM
IC50 > 20 μM
1
D
IC50 = 45 nM
H
N
O
H2N
N
N
N
N
O
O
O
10*
IC50 > 5 μM
11*
IC50 > 20 μM
12
* racemates with isobutyl
(from L-leucine) in zone C
IC50 > 20 μM
Figure 4. SAR overview of Zone A, 20 compounds prepared.
The SAR strategy was based on the modification of four
zones, A–D, of the azetidinone molecule as depicted in
Figure 4. A limited SAR explored effects of substitution
in Zone A. For chemical accessibility, the dioxolane ring
was replaced with isobutyl in many of the analogs (i.e.,
L-leucine benzyl ester was used in the 2 + 2 reaction).
The affinity of the parent isobutyl analog, 18, was
39 nM (see Fig. 6, below). Several alterations afforded
a variety of azetidinones with a significant loss of V1a
binding activity as represented in Figure 4. Similarly, a
limited SAR at Zone B demonstrated that several mod-
ifications afforded derivatives with reduced V1a binding
activity. Interestingly, replacing the phenyl with the 2-
furyl substitution afforded a compound, 14, with com-
parable activity, V1a IC50 = 2 nM (Fig. 5).
the ester linkage. As an intermediate, the trimethylsilyl
ester derivative, 19, was prepared. Despite having equiv-
alent bulk and hydrophobicity to the benzyl group, the
trimethylsilyl substitution resulted in reduced activity
(V1a IC50 = 405 nM), indicating that the shape of the
benzyl group was important. The trimethylsilyl ester,
in turn, was transformed into the acid 20, and subse-
quently coupled with benzylamine to afford the benzyl
amide, 21 (V1a IC50 = 43 nM). Thus, the ester function-
ality of 1 could be converted to the corresponding
amide, 21, without loss of binding affinity (V1a
IC50 = 45 nM vs V1a IC50 = 43 nM, respectively, see
Fig. 7).
At this point, the next step for this project was to find a
lead structure that combined high receptor affinity
(IC50 < 10 nM) with useful brain levels (>10· the recep-
tor affinity). Although 6 had good V1a binding affinity
(IC50 = 5 nM), it provided minimal brain penetration
in dogs treated orally with 10 mg/kg (dog brain concen-
tration of 6, 10 nM at 4 h). In an effort to find an amide
substitution that would enhance oral absorption and
CNS penetration, the L-leucyl platform was constructed
(Fig. 8). Although not optimal for affinity, this platform
was used as the basis for SAR exploration of Zone D
because of its stability under conditions used for synthesis.
The Zone D explorations had two objectives: to find the
optimal substitution pattern for the benzylamide group,
and to find other groups that would be compatible with
high affinity, especially groups with charged functions
Zones C and D emerged as the most important points of
structural modification. Removal of the Zone C substi-
tuent, as illustrated by the simple benzyl glycine deriva-
tive 5, resulted in 10-fold loss of V1a activity,
IC50 = 400 nM. Interestingly, acylation of 5 with base
and pivaloyl chloride afforded 6 with significantly en-
hanced potency (V1a IC50 = 5 nM). Several Zone C
modifications and attendant human IC50 values are
shown in Figure 6.
In 1, Zone D is occupied by the benzyl ester. The ester
functionality was considered a potential metabolic weak
point, so the first priority was to ascertain whether a
more stable group such as amide could substitute for
O
O
A
B
H
N
O
O
O
O
N
C
N
N
N
O
O
O
O
O
13
14
15
IC50 = 2 nM
IC50 > 500 nM
IC50 > 20 μM
methyldioxolane
in zone C
6
D
single pivaloyl
diastereomer
L-isobutyl
in zone C
IC50 = 5 nM
Figure 5. SAR overview of Zone B, 3 compounds prepared.

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C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
O
O
A
B
O
O
N
O
N
N
O
O
O
O
N
C
O
BzO2C
BzO2C
6
16
O
IC50 = 5 nM
IC50 = 100 nM
1
D
IC50 = 45 nM
OH
N
N
N
O
O
O
BzO2C
BzO2C
BzO2C
5
17
18
IC50 = 39 nM
IC50 = 400 nM
IC50 = 10 nM
Figure 6. SAR overview of Zone C, 35 compounds prepared.
19
N
O
IC50 = 405 nM
O
O
Me3Si(CH2)2O
A
B
O
N
O
O
O
20
N
C
O
N
IC50 > 5 μM
O
HO2C
O
1
D
IC50 = 45 nM
21
N
IC50 = 43 nM
O
BzNH
O
Figure 7. Acceptability of benzylamide in Zone D.
O
O
24
N
N
IC50 = 128 nM
N
O
N
25
IC50 = 1 μM
Cl
N
R
O
26
L-leucyl platform
IC50 = 40 nM
N
Cl
22
IC50 = 78 nM
N
N
27
IC50 = 41 nM
CF3
N
23
N
28
IC50 = 54 nM
N
IC50 = 262 nM
Cl
Figure 8. SAR overview of Zone D.
that would improve water solubility. Rapid solution-
phase amide syntheses mediated by solid phase carbodii-
mide afforded over 50 amides,22 some of which are
illustrated in Figure 8.
A small substituent at the 3-position of the benzylamide
(e.g., Cl or CF3) was found to provide optimal affinity.
Among groups other than benzyl, piperidylpiperidine
and piperidyl-ethyl-piperidine provided relatively good

C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
2059
O
O
O
O
N
O
N
O
O
N
N
O
N
O
O
N
6
22
IC50 = 5 nM
IC50 = 78 nM
O
O
10 nM dog brain
100 nM dog brain
N
O
O
N
29
IC50 = 85 nM (predicted 10 nM)
N
O
N
Figure 9. Non-additivity of the Zone C pivaloyl and Zone D piperidyl-ethyl-piperidine substitutions.
affinities. These groups would be positively charged at
physiological pH, potentially leading to improved solu-
bility. Interestingly, the piperidylpiperidine group was
previously shown to provide optimal in vivo activity in
an NK-1 clinical candidate.23
ticals (later renamed Azevan), a start-up company creat-
ed for this purpose. The initial strategy was to construct
a scaffold with aminomalonic acid replacing L-leucine as
depicted in Figure 10. If the piperidyl-ethyl-piperidine
binds into Zone C according to the previous hypothesis,
the binding pockets in Zones C and D should each tol-
erate a side chain anchored by an amide coupling, allow-
ing the piperidyl-ethyl-piperidinamide and benzylamide
to be present simultaneously. With the correct stereo-
chemistry, each group should be able to bind to its pre-
ferred pocket. The relevant aminomalonate platform
was synthesized as illustrated in Scheme 2. Gratifyingly,
33 showed a dramatic enhancement of human V1a bind-
ing affinity (V1a Ki = 1.17 nM, Fig. 11).
The piperidyl-ethyl-piperidine 22 was evaluated in dogs
dosed orally at 10 mg/kg. Its brain level at 4 h post-ad-
ministration was 100 nM, 10-fold better than 6 (see
Fig. 9). Because the isobutyl substitution in Zone C
was known to be suboptimal for V1a affinity, the corre-
sponding compound with pivaloyl in Zone C was syn-
thesized. Based on previous results, the pivaloyl group
was expected to provide a roughly 8-fold boost in affin-
ity, resulting in a predicted affinity of ꢀ10 nM for the
combination. Instead, affinity was 85 nM (Fig. 9).
Even though there was significant improvement in bind-
ing affinity in the aminomalonyl platform azetidinones
(Fig. 11), the compounds could only be obtained as dia-
stereomeric mixtures, as a consequence of planarization
of the malonate methine on dissociation of its acidic
hydrogen. Consequently the L-aspartyl platform was
prepared in the hope that this would retain the V1a
binding affinity while obviating the issue of the labile
methine hydrogen and the attendant diastereomeric
mixture. These synthetic transformations are illustrated
in Scheme 3. The most potent compound synthesized,
37, had a V1a affinity comparable to that of 33 from
the aminomalonyl platform (Fig. 12).
In searching for an explanation of the non-additivity
seen in 29, it occurred to us that the binding mode of
the molecule may have changed when benzyl was re-
placed with piperidyl-ethyl-piperidine. The two groups
are after all completely different in shape and charge.
With a proximally branched alkyl structure, the piper-
idyl-ethyl-piperidine group actually bears more resem-
blance to a typical Zone C substituent, such as
methyldioxolane, pivaloyl, or isobutyl. We conjectured
that the piperidyl-ethyl-piperidine bound into the Zone
C pocket of the receptor, forcing the pivaloyl group into
the free aqueous phase or into an unfavorable interac-
tion with the Zone D pocket of the receptor. By this log-
ic, reintroducing the benzylamide group in place of the
pivaloyl should reoptimize binding in the Zone D pock-
et, creating a large boost in affinity. At this point, the
vasopressin project was deprioritized, so confirmation
of this hypothesis was put on hold pending completion
of a successful out-licensing campaign.
Theorizing that the D-aspartyl platform would be more
resistant to in vivo enzymatic hydrolysis, we prepared
the platform with a D-aspartyl group in Zones C and
D (see Scheme 4). Although in general the D-platforms
tended to be less active than the L-platforms (see the
D-glutamyl platform, below), this was less pronounced
for the D-aspartyl platform. In the case of 41 (V1a Ki
1.49 nM, Fig. 13), affinity of the D-aspartyl compound
was actually slightly better than that of the correspond-
ing L-aspartyl analog (42, V1a Ki = 1.89 nM). It is
After a four-year search for a partner, the program was
reconstituted as a joint venture with Serenix Pharmaceu-

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C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
O
O
O
O
N
O
N
O
O
O
N
N
N
O
O
O
6
29
IC50 = 85 nM
IC50 = 5 nM
N
O
O
N
O
O
N
N
N
N
O
H
Figure 10. Rationale for aminomalonate platform.
O
N
O
O
O
F3C
NH2
N
O
O
N
O
O
H
N
THF
N
H
OtBu
O
H
O
CCl3
O
31
30
OtBu
F3C
HCO2H
O
H
N
O
O
O
N
2HCl
N
O
N
N
O
O
N
H
N
H
O
NEt3
HOAT
EDC, HCl
CH2Cl2
H
N
N
O
H
O
OH
F3C
N
32
33
F3C
HNR1R2
HOAT
O
O
EDC, HCl
CH2Cl2
N
O
O
N
N
H
R1R2N
O
F3C
Scheme 2. Synthesis of 33 and analogs.
possible that the piperidylpiperidine side-chain may be
able to adapt its conformation to match the receptor
pocket in a unique way—a possibility that may also ex-
plain its superior pharmacokinetic properties in this ser-
ies (vide infra) and the NK-1 series cited previously.23
The same reasons that led to the investigation of the
L- and D-aspartyl platforms also resulted in the prepara-
tion of a range of compounds derived from L- and D-glu-
tamic acids. The syntheses of these L- and D-glutamyl
platforms are also illustrated in Schemes 3–5. The

C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
2061
33:
37:
R1 = H
R2 =
R1 = H
R2 =
O
O
O
O
O
N
N
N
N
O
N
O
N
H
O
O
N
N
R2
R2
R3 =
R3 =
N
H
N
R1
R3
R1
R3
H
CF
CF
O
3
Ki = 1.17 nM
10 Compounds prepared,
5 with Ki < 20 nM
Ki = 1.13 nM
61 Compounds prepared,
5 with Ki < 20 nM
Figure 11. D,L-Aminomalonyl platform and attendant V1a affinity.
Figure 12. L-Aspartyl platform and attendant V1a affinity.
corresponding V1a binding affinities are shown (see
Figs. 14 and 15). For these two series, the most active
compounds were found to be L-glutamate analogs, with
the D-glutamate versions being typically about 20-fold
less active.
the azetidine N. Thus, it appears that optimal activity
requires a positively charged group at a fixed distance
from the azetidinone core. It is possible that the basic
nitrogen binds to the same site as the guanidinium of
arginine–vasopressin.
Five of the compounds with Ki < 10 nM24 were tested
for plasma and brain levels in rats after oral dosing
(20 mg/kg). Azetidinone 22 was used as a comparator.
In this screening experiment, three compounds were
dosed per animal, and plasma and brain levels were
measured by LC/MS. Compound 41 showed the highest
levels in both plasma and brain; however, several-fold
lower levels were seen in follow-up experiments where
It is of interest that the highest affinity in the glutamyl
platform was seen with the 4-cyclohexylpiperazine side
chain, which is identical to the piperidylpiperidine side
chain that gave optimal activity in the aspartyl platform,
except that the basic amino group has been moved one
bond closer, offsetting the additional methylene in the
glutamyl moiety. In both of these examples, as well as
in 37, the positively charged group is 8 bonds away from
i or
ii
O
O
H2, Pd/C
MeOH
O
O
O
O
NH
NH
R
NR1R2
tBuO
n
tBuO
O
n
O
O
34
O
NH2
NR1R2
O
tBuO
35
N
n
Cl
O
CO2H
CH2Cl2
DMF
O
O
Cl
CHO
O
O
O
N
O
O
N
O
COCl
+
N
CH2Cl2 / MgSO4
O
N
NR1R2
n
NR1R2
O
tBuO
n
tBuO
O
4
36
HCO2H
O
N
O
O
O
R3R4NH
HOAT
EDC,HCl
CH2Cl2
CF3
37: n = 1
-NR1R2 =
N
O
H
O
N
O
N
N
O
NR1R2
NR1R2
H
n
n
-NR3R4 =
N
N
O
O
R3R4N
HO
Scheme 3. Syntheses of L-aspartic and L-glutamic acid derivatives. R = –OSu. Reagents: (i) HNR1R2/THF; R = –OH, (ii) HNR1R2/HOBT/EDC,
HCl/CH2Cl2.

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C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
R1R2NH
HOAT
EDC,HCl
CH2Cl2
O
O
H2, Pd/C
MeOH
O
O
O
O
NH
n
NH
OH
NR1R2
tBuO
tBuO
O
n
O
O
38
O
NH2
NR1R2
O
tBuO
N
n
Cl
O
CO2H
CH2Cl2
DMF
O
39
O
Cl
CHO
1
O
O
O
N
O
O
N
O
COCl
+
N
CH2Cl2 / MgSO4
O
N
NR1R2
n
NR1R2
O
tBuO
n
tBuO
O
4
40
HCO2H
O
N
O
O
O
R3R4NH
HOAT
EDC,HCl
CH2Cl2
CF3
41: n =1
-NR1R2 =
N
O
H
O
N
O
N
N
O
NR1R2
NR1R2
n
n
N
N
-NR3R4 =
O
O
R3R4N
HO
Scheme 4. Syntheses of D-aspartic and D-glutamic acid derivatives.
41:
52, the (S)-a-methylbenzyl diastereomer of 50, was
600-fold less potent (V1a Ki = 179 nM). Interestingly,
53, the trifluoromethyl analog of 50, had reduced V1a
binding affinity (see Fig. 16), suggesting that the a-meth-
yl substitution shifted the binding mode of the adjacent
phenyl ring. Azetidinone 54, the N-methyl derivative of
50, likewise had poor activity (V1a Ki = 9.8 nM), indi-
cating that the N-methyl and a-methyl modifications
also were incompatible.
O
N
N
R1 =
O
O
N
O
R2 = H
R3 =
N
R2
N
R1
R3
H
CF3
O
Ki = 1.49 nM
18 Compounds prepared,
6 with Ki < 20 nM
Plasma levels for 50 and 51 were roughly double those of
41, to some degree confirming the original motivation
for the construction of these two compounds. Cmax val-
ues were 100 ng/ml and 160 ng/ml, respectively, com-
pared to 50–80 ng/ml for 41 (Fig. 17). Plasma Tmax
values for 50 and 51 were 4 h. Plasma T1/2 values for
50 and 51 were 2.5 h and 4 h, respectively. The brain lev-
els of 50 and 51 exceeded their Ki values by approxi-
mately 100-fold; brain Cmax values of 50 and 51 were
20 ng/ml and 48 ng/ml, respectively, equivalent to
28 nM and 62 nM. Brain T1/2 values of ꢀ6 h for both
compounds suggest the possibility of once-a-day dosing.
Figure 13. D-Aspartyl platform and attendant V1a affinity.
41 was dosed alone, suggesting that the initial higher lev-
els were due in part to co-dosing with 22. These results
(plasma Cmax 50–80 ng/ml in two separate experiments)
were considered encouraging but fell somewhat short of
expectations for a clinical candidate.
Theorizing that facile enzymatic cleavage of the benzyla-
mide bond in Zone D reduced levels of 41, we prepared
50 (SRX246) and 51 (SRX251) (see Fig. 16 for structures
and Scheme 4 for synthetic pathway). It was hoped that
these molecules, the (R)-a-methylbenzyl and the N-
methyl analogs of 41, respectively, would be more stable
to enzymatic hydrolysis due to steric hindrance near the
amide bond. Fortuitously, 50 and 51 proved to possess
even higher in vitro V1a affinity than 41. Compound
The binding affinity of all the reported compounds to
other members of the vasopressin receptor family was
also tested using cell lines that expressed human V1b
and V2. The methods for human V1b and V2 binding
assay are similar to that for V1a screening, namely the

C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
2063
R1R2NH
HOAT
EDC, HCl
CH2Cl2
O
NH2
O
H2, Pd/C
MeOH
O
OtBu
O
O
O
R1R2N
44
n
O
NH
n
NH
O
OtBu
OtBu
HO
R1R2N
CH2Cl2
MgSO4
n
O
O
43
O
O
CHO
N
CO2H
CH2Cl2
DMF
O
Cl
1
Cl
O
O
N
O
O
O
O
O
N
O
N
O
COCl
+
O
OH
O
N
N
CH2Cl2
NET3
HCO2H
O
N
OtBu
n
n
OtBu
R1R2N
O
O
n
O
R1R2N
R1R2N
4
45
Scheme 5. Synthesis of additional L-glutamic acid derivatives.
46:
whole cell-based competitive binding assay, which is de-
scribed in Section 4. The IC50 values of most Azevan
compounds proved to be greater than 1 lM or 10 lM
for both the V1b and V2 human receptor cell lines.
R1 = H
O
O
N
N
N
O
R2 =
R3 =
O
N
R2
N
H
R1
R3
3. Conclusions
CF
O
3
A novel series of vasopressin V1a antagonists has been
designed from the unique monocyclic azetidinone plat-
form. Subnanomolar affinities at the human V1a recep-
tor have been achieved. On oral dosing, two members of
the series, 50 (SRX246) and 51 (SRX251), reached brain
levels ꢀ100 times their in vitro receptor affinities. The
candidate molecules are being further developed for hu-
man clinical evaluation.
Ki = 1.17 nM
>150 Compounds prepared,
>60 with Ki < 20 nM
47:
48:
R1 = H
R1 = H
R2 =
N
N
N
N
R2 =
R3 =
O
O
R3 =
N
H
N
H
CF3
CF
O
O
4. Experimental
4.1. Chemistry
Ki = 1.45 nM
Ki = 0.27 nM
Figure 14. L-Glutamyl platform and attendant V1a affinity.
The early experimental work that was initially per-
formed at Eli Lilly (preparation and characterization
of LY307174, etc.) is described in Ref. 22.
49:
4.1.1. (4(S)-Phenyloxazolidin-2-on-3-yl)acetyl chloride
(4). This material was not produced in one batch but
rather prepared as required for each ‘chiral Staudinger
2 + 2 cycloaddition reaction.’ Typically, a solution of
1 g (4.52 mmol) of (4(S)-phenyloxazolidin-2-on-3-
yl)acetic acid (Evans, U.S. Patent No. 4,665,171) and
0.51 mL (5.88 mmol) of oxalyl chloride in 150 mL of
dichloromethane was treated with a catalytic amount
of anhydrous dimethylformamide (85 lL/mequiv of
acetic acid derivative) resulting in vigorous gas
evolution. After 45 min, all gas evolution had ceased
O
O
R1 =
N
N
O
N
O
R2 = H
R3 =
N
R2
R1
R3
N
H
CF3
O
7 Compounds prepared,
3 with Ki < 20 nM
Ki = 5.84 nM
Figure 15. D-Glutamyl platform and attendant V1a affinity.

2064
C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
O
O
O
O
O
O
N
O
N
O
N
O
N
N
N
O
O
O
H
N
H
N
N
N
N
N
N
N
N
O
O
O
F3C
F3C
53, Ki = 1.27 nM
50, Ki = 0.3 nM
51, Ki = 0.66 nM
Figure 16. Structures and human V1a binding affinities of 50, 51, and 53.
plasma
ng/
brain
ng/
mL
200
150
100
50
5
4
3
2
1
0
5
4
3
2
1
0
mL
5
4
3
2
1
0
60
50
40
30
20
10
0
7
6
5
4
3
2
1
0
h
h
h
h
0
Cmax
Tmax
T1/2
Cmax
Tmax
1/2
SRX246
SRX251
Figure 17. Oral pharmacokinetics of SRX246 and SRX251 in rats.
and the reaction mixture was concentrated under re-
duced pressure to provide the title compound as an
off-white solid after drying for 2 h under vacuum. Com-
pound 4 was used without purification for the ‘chiral
Staudinger [2 + 2] cycloaddition reaction.’
4.1.2.2. N-Benzyloxycarbonyl-L-aspartic acid b-tert-
butyl ester a-[4-(2-phenylethyl)]piperazinamide (34b, 34
with HNR1R2 = 4-(phenylethyl)piperazine and n = 1).
Use of N-benzyloxycarbonyl-L-aspartic acid b-tert-butyl
ester a-N-hydroxysuccinimide ester (5.0 g, 12 mmol, Ad-
vanced ChemTech) and 4-(phenylethyl)piperazine 2.27 mL
(11.9 mmol) gave 5.89 g (quantitative yield) of 34b as a
white solid; 1H NMR (CDCl3) d 1.40 (s, 9H); 2.45–
2.80 (m, 10H); 3.50–3.80 (m, 4H); 4.87–4.91 (m, 1H);
5.08 (s, 2H); 5.62–5.66 (m, 1H); 7.17–7.33 (m, 10H).
4.1.2. General procedure for an amide formation from an
activated ester derivative
4.1.2.1. N-Benzyloxycarbonyl-L-aspartic acid b-tert-
butyl ester a-(3-trifluoromethyl)benzylamide (34a, 34 with
HNR1R2 = 3-(trifluoromethyl)benzylamine and n = 1). A
solution of N-benzyloxycarbonyl-L-aspartic acid b-tert-
butyl ester a-N-hydroxysuccinimide ester (1.95 g,
4.64 mmol, Advanced ChemTech) in 20 mL of dry tetra-
hydrofuran was treated with 0.68 mL (4.74 mmol) of 3-
(trifluoromethyl)benzylamine. Upon completion of the
reaction as monitored by TLC (60:40 hexanes/ethyl ace-
tate), the solvent was evaporated, and the resulting oil
was partitioned between dichloromethane and a saturat-
ed aqueous solution of sodium bicarbonate. The organic
layer was dried over magnesium sulfate, filtered, and
evaporated to give 2.23 g (quantitative yield) of the title
compound as a white solid; 1H NMR (CDCl3) d 1.39 (s,
9H); 2.61 (dd, J = 6.5 Hz, J = 17.2 Hz, 1H); 2.98 (dd,
J = 3.7 Hz, J = 17.0 Hz, 1H); 4.41 (dd, J = 5.9 Hz,
J = 15.3 Hz, 1H); 4.50–4.57 (m, 2H); 5.15 (s, 2H);
5.96–5.99 (m, 1H); 6.95 (s, 1H); 7.29–7.34 (m, 5H);
7.39–7.43 (m, 2H); 7.48–7.52 (m, 2H).
4.1.2.3. N-Benzyloxycarbonyl-L-glutamic acid c-tert-
butyl ester a-(3-trifluoromethyl)benzylamide (34c, 34 with
HNR1R2 = 3-(trifluoromethyl)benzylamine and n = 2).
Use of N-benzyloxycarbonyl-L-glutamic acid b-tert-bu-
tyl ester a-N-hydroxysuccinimide ester (4.83 g,
11.1 mmol, Advanced ChemTech) and 3-(trifluorometh-
yl)benzylamine 1.63 mL (11.4 mmol) gave 5.41 g (98%)
1
of 34c as a white solid; H NMR (CDCl3) d 1.40 (s,
9H); 1.88–1.99 (m, 1H); 2.03–2.13 (m, 1H); 2.23–2.33
(m, 1H); 2.38–2.47 (m, 1H); 4.19–4.25 (s, 1H); 4.46–4.48
(m, 2H); 5.05–5.08 (m, 2H); 5.67–5.72 (m, 1H); 7.27–
7.34 (m, 5H); 7.39–7.43 (m, 2H); 7.48–7.52 (m, 2H).
4.1.3. General procedure for amide formation from a
carboxylic acid
4.1.3.1. N-Benzyloxycarbonyl-D-aspartic acid b-tert-
butyl ester a-(3-trifluoromethyl)benzylamide (38a, 38 with
HNR1R2 = 3-(trifluoromethyl)benzylamine and n = 1).
A solution of 1 g (2.93 mmol) of N-benzyloxycarbonyl-
D-aspartic acid b-tert-butyl ester monohydrate
The following compounds were obtained according to
this procedure:

C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
2065
(Novabiochem) in 3–4 mL of dichloromethane was treat-
ed by sequential addition of 0.46 mL (3.21 mmol)
of 3-(trifluoromethyl)benzylamine, 0.44 g (3.23 mmol) of
1-hydroxy-7-benzotriazole, and 0.62 g (3.23 mmol) of
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydro-
chloride. After at least 12 h at ambient temperature or
until complete as determined by thin layer chromatogra-
phy (95:5 dichloromethane/methanol eluent), the
reaction mixture was washed sequentially with a saturat-
ed aqueous sodium bicarbonate solution and with
distilled water. The organic layer was dried over magne-
sium sulfate, filtered, and evaporated to give 1.41 g
4.1.3.5. N-Benzyloxycarbonyl-D-aspartic acid c-tert-
butyl ester a-[N-methyl-N-(3-trifluoromethylbenzyl)]amide
(38e, 38 with HNR1R2 = N-methyl-N-(3-trifluoromethyl-
benzyl)amine and n = 1). Use of N-benzyloxycarbonyl-D-
aspartic acid c-tert-butyl ester (0.303 g, 0.89 mmol,
Novabiochem) and 0.168 g (0.89 mmol,) of N-methyl-
N-(3-trifluoromethylbenzyl)amine gave 0.287 g (65%)
1
of 38e as a white solid; H NMR (CDCl3) d 1.40 (s,
9H); 2.55 (dd, J = 5.8 Hz, J = 15.8 Hz, 1H); 2.81 (dd,
J = 7.8 Hz, J = 15.8 Hz, 1H); 4.10 (s, 3H); 4.25 (d,
J = 15.0 Hz, 1H); 4.80 (d, J = 15.5 Hz, 1H); 5.01–5.13
(m, 3H); 5.52–5.55 (m, 1H); 7.25–7.52 (m, 10H).
1
(quantitative yield) of 38a as a white solid; H NMR
(CDCl3) d 1.39 (s, 9H); 2.61 (dd, J = 6.5 Hz, J =
17.2 Hz, 1H); 2.98 (dd, J = 4.2 Hz, J = 17.2 Hz, 1H);
4.41 (dd, J = 5.9 Hz, J = 15.3 Hz, 1H); 4.50–4.57 (m,
2H); 5.10 (s, 2H); 5.96–6.01 (m, 1H); 6.91–7.00 (m,
1H); 7.30–7.36 (m, 5H); 7.39–7.43 (m, 2H); 7.48–7.52
(m, 2H).
4.1.3.6. N-Benzyloxycarbonyl-D-aspartic acid b-tert-
butyl ester a-[(S)-1-(3-trifluoromethylphenyl)ethyl]amide
(38f, 38 with HNR1R2 = (S)-1-(3-trifluoromethylphen-
yl)ethylamine and n = 1). Use of N-benzyloxycarbonyl-
D-aspartic acid b-tert-butyl ester monohydrate (Nova-
biochem) (84 mg, 0.25 mmol) and 47 mg of (S)-1-(3-tri-
fluoromethylphenyl)ethylamine
gave
122 mg
1
The following compounds were obtained according to
this procedure:
(quantitative yield) of 38f as a white solid; H NMR
(CDCl3) d 1.38 (s, 9H); 1.43 (d, J = 7.0 Hz, 3H); 2.59
(dd, J = 6.7 Hz, J = 17.3 Hz, 1H); 2.93 (dd, J = 4.1 Hz,
J = 17.3 Hz, 1H); 4.49–4.53 (m, 1H); 5.05–5.28 (m,
3H); 5.91–5.95 (m, 1H); 6.84–6.87 (m, 1H); 7.29–7.52
(m, 9H).
4.1.3.2. N-Benzyloxycarbonyl-D-aspartic acid b-tert-
butyl ester a-(2-fluoro-3-trifluoromethyl)benzylamide
(38b, 38 with HNR1R2 = (2-fluoro-3-trifluoromethyl)ben-
zylamine and n = 1). Use of N-benzyloxycarbonyl-D-
aspartic acid b-tert-butyl ester monohydrate (Novabio-
chem) (0.25 g, 0.73 mmol) and 0.12 mL of (2-fluoro-3-
trifluoromethyl)benzylamine gave 0.365 g (quantitative
yield) of 38b as a white solid; 1H NMR (CDCl3) d
1.38 (s, 9H); 2.59 (dd, J = 6.5 Hz, J = 17.0 Hz, 1H);
2.95 (dd, J = 4.3 Hz, J = 17.0 Hz, 1H); 4.46–4.56 (m,
3H); 5.11 (s, 2H); 5.94–5.96 (m, 1H); 7.15 (t,
J = 8.0 Hz, 1H); 7.30–7.36 (m, 5H); 7.47–7.52 (m, 2H).
4.1.3.7. N-Benzyloxycarbonyl-D-aspartic acid b-tert-
butyl ester a-[(R)-1-(3-trifluoromethylphenyl)ethyl]amide
(38g, 38 with HNR1R2 = (R)-1-(3-trifluoromethylphen-
yl)ethylamine and n = 1). Use of N-benzyloxycarbonyl-
D-aspartic acid b-tert-butyl ester monohydrate (Nova-
biochem) (150 mg, 0.44 mmol) and 83 mg of (R)-1-(3-
trifluoromethylphenyl)ethylamine gave 217 mg (quanti-
1
tative yield) of 38g as a white solid; H NMR (CDCl3)
d 1.38 (s, 9H); 1.43 (d, J = 7.0 Hz, 3H); 2.54 (dd,
J = 7.4 Hz, J = 17.3 Hz, 1H); 2.87 (dd, J = 4.0 Hz,
J = 17.3 Hz, 1H); 4.47–4.51 (m, 1H); 5.01–5.16 (m,
3H); 5.92–5.96 (m, 1H); 6.90–6.94 (m, 1H); 7.26–7.49
(m, 9H).
4.1.3.3. N-Benzyloxycarbonyl-D-aspartic acid b-tert-
butyl ester a-[(S)-a-methylbenzyl]amide (38c, 38 with
HNR1R2 = (S)-a- methylbenzylamine and n = 1). Use of
N-benzyloxycarbonyl-D-aspartic acid b-tert-butyl ester
monohydrate (Novabiochem) (0.25 g, 0.73 mmol) and
0.094 mL of (S)-a-methylbenzylamine gave 0.281 g
(90%) of 38c as a white solid; 1H NMR (CDCl3) d
1.41 (s, 9H); 1.44 (d, J = 7.0 Hz, 3H); 2.61 (dd,
J = 7.0 Hz, J = 17.0 Hz, 1H); 2.93 (dd, J = 4.0 Hz,
J = 17.5 Hz, 1H); 4.50–4.54 (m, 1H); 5.04–5.14 (m,
3H); 5.94–5.96 (m, 1H); 6.76–6.80 (m, 1H); 7.21–7.37
(m, 10H).
4.1.3.8. N-Benzyloxycarbonyl-D-glutamic acid c-tert-
butyl ester a-(3-trifluoromethyl)benzylamide (38h, 38 with
HNR1R2 = 3-(trifluoromethyl)benzylamine and n = 2).
Use of N-benzyloxycarbonyl-D-glutamic acid c-tert-bu-
tyl ester (1.14 g, 3.37 mmol) and 0.53 mL (3.70 mmol,
Novabiochem) of 3-(trifluoromethyl)benzylamine gave
1
1.67 g (quantitative yield) of 38h as a white solid; H
NMR (CDCl3) d 1.40 (s, 9H), 1.89–1.97 (m, 1H);
2.04–2.12 (m, 1H); 2.24–2.31 (m, 1H); 2.39–2.46 (m,
1H); 4.19–4.24 (s, 1H); 4.45–4.48 (m, 2H); 5.03–5.09
(m, 2H); 5.71–5.74 (m, 1H); 6.83–6.88 (m, 1H); 7.26–
7.34 (m, 5H); 7.40–7.43 (m, 2H); 7.49–7.52 (m,
2H).13C NMR (CDCl3) d 27.86, 27.99, 31.73, 54.51,
4.1.3.4. N-Benzyloxycarbonyl-D-aspartic acid b-tert-
butyl ester a-[(R)-a-methylbenzyl]amide (38d, 38 with
HNR1R2 = (R)-a-methylbenzylamine and n = 1). Use of
N-benzyloxycarbonyl-D-aspartic acid b-tert-butyl ester
monohydrate (Novabiochem) (0.25 g, 0.73 mmol) and
0.094 mL of (R)-a-methylbenzylamine gave 0.281 g
3
67.11, 81.19, 122.90, 124.22 (q, JC–F, 4.0 Hz, 1C),
1
3
124.31 (q, JC–F, 3.7 Hz, 1C), 125.06, 128.03, 128.22,
(90%) of 38d as a white solid; H NMR (CDCl3) d 1.38
1
128.52, 129.17, 130.88, 130.98 (q, JC–F, 32.3 Hz, CF3),
136.07, 139.02, 171.43, 172.92.
(s, 9H); 1.43 (d, J = 6.9 Hz, 3H); 2.54 (dd, J = 7.3 Hz,
J = 17.2 Hz, 1H); 2.87 (dd, J = 4.1 Hz, J = 17.2 Hz,
1H); 4.46–4.50 (m, 1H); 4.99–5.15 (m, 3H); 5.92–5.96
(m, 1H); 6.78–6.82 (m, 1H); 7.21–7.33 (m, 10H). 13C
NMR (CDCl3) d 21.99, 27.95, 37.58, 50.03, 50.92, 67.13,
81.87, 125.95, 126.72, 128.05, 128.21, 128.52, 128.58,
129.07, 136.08, 142.86, 155.93, 169.34, 171.31.
4.1.3.9. N-Benzyloxycarbonyl-L-glutamic acid a-tert-
butyl ester c-(4-cyclohexyl)piperazinamide: (43a, 43 with
HNR1R2 = 1-cyclohexylpiperazine and n = 2). Use of
N-benzyloxycarbonyl-L-glutamic acid a-tert-butyl ester

2066
C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
(1.36 g, 4.03 mmol) and 0.746 g (4.43 mmol) of 1-cyclo-
hexylpiperazine gave 1.93 g (98%) of 43a as a white sol-
id; 1H NMR (CDCl3) d 1.02–1.12 (m, 5H); 1.43 (s, 9H),
1.60–1.64 (m, 1H); 1.80–1.93 (m, 5H); 2.18–2.52 (m,
8H); 3.38–3.60 (m, 4H); 4.20–4.24 (m, 1H); 5.03–5.13
(m, 2H); 5.53–5.57 (m, 1H); 7.28–7.34 (m, 5H).
N-benzyloxycarbonyl-D-aspartic acid b-tert-butyl ester
a-(2-fluoro-3-trifluoromethyl)benzylamide 38b (0.36 g,
0.72 mmol) gave 0.256 g (92%) of 39b as an off-white
solid; 1H NMR (CDCl3) d 1.39 (s, 9H); 2.50 (br s,
2H); 2.74 (dd, J = 7.0 Hz, J = 16.5 Hz, 1H); 2.86 (dd,
J = 4.8 Hz, J = 16.8 Hz, 1H); 3.89 (br s, 2H); 4.47–4.57
(m, 2H); 7.16 (t, J = 7.8 Hz, 1H); 7.48 (t, J = 7.3 Hz,
1H); 7.56 (t, J = 7.3 Hz, 1H); 7.97–8.02 (m, 1H).
4.1.4. General procedure for hydrogenation of a benzyl-
oxycarbonyl amine
4.1.4.1. L-Aspartic acid b-tert-butyl ester a-(3-trifluo-
romethyl)benzylamide (35a, 35 with HNR1R2 = 3-(tri-
fluoromethyl)benzylamine and n = 1). A suspension of
2.23 g (4.64 mmol) of N-benzyloxycarbonyl-L-aspartic
acid b-tert-butyl ester a-(3-trifluoromethyl)benzylamide
34a and palladium (5% wt. on activated carbon,
0.642 g) in 30 mL of methanol was held under an atmo-
sphere of hydrogen until complete conversion as deter-
mined by thin layer chromatography (95:5
dichloromethane/methanol eluent). The reaction was fil-
tered to remove the palladium over carbon and the fil-
trate was evaporated to give 1.52 g (96%) of 7a as an
4.1.4.6. D-Aspartic acid b-tert-butyl ester a-[(S)-a-
methyl]benzylamide (39c, 39 with HNR1R2 = (S)-a-methyl-
benzylamine and n = 1). Use of N-benzyloxycarbonyl-D-
aspartic acid b-tert-butyl ester a-[(S)-a-methylben-
zyl]amide (0.275 g, 0.65 mmol) 38c gave 0.17 g (90%)
1
of 39c as an off-white solid; H NMR (CDCl3) d 1.40
(s, 9H); 1.47 (d, J = 6.9 Hz, 3H); 1.98 (br s, 2H); 2.49
(dd, J = 7.9 Hz, J = 17.7 Hz, 1H); 2.83 (dd, J = 3.6 Hz,
J = 16.7 Hz, 1H); 3.69 (br s, 1H); 4.99–5.10 (m, 1H);
7.19–7.33 (m, 5H); 7.65–7.68 (m, 1H).
4.1.4.7. D-Aspartic acid b-tert-butyl ester a-[(R)-a-
methylbenzyl]amide (39d, 39 with HNR1R2 = (R)-a-methyl-
benzylamine and n = 1). Use of N-benzyloxycarbonyl-D-
aspartic acid b-tert-butyl ester a-[(R)-a-methylben-
zyl]amide (0.273 g, 0.64 mmol) 38d gave 0.187 g (quanti-
tative yield) of 39d as an off-white solid; 1H NMR
(CD3OD) d 1.40 (s, 9H); 1.46 (d, J = 7.0 Hz, 3H); 2.49
(dd, J = 7.0 Hz, J = 16.5 Hz, 1H); 2.61 (dd, J = 5.5 Hz,
J = 16.0 Hz, 1H); 3.62 (dd, J = 6.0 Hz, J = 7.0 Hz,
1H); 4.98 (dd, J = 7.0 Hz, J = 14.0 Hz, 1H); 7.20–7.23
(m, 1H); 7.27–7.34 (m, 4H); 13C NMR (CD3OD) d
22.45, 28.31, 41.38, 50.17, 52.92, 82.17, 127.13, 128.09,
129.53, 144.96, 172.14, 175.11.
1
off-white solid; H NMR (CDCl3) d 1.42 (s, 9H); 2.26
(br s, 2H); 2.63–2.71 (m, 1H); 2.82–2.87 (m, 1H); 3.75–
3.77 (m, 1H); 4.47–4.50 (m, 2H); 7.41–7.52 (m, 4H);
7.90 (br s, 1H).
The following compounds were obtained according to
this procedure:
4.1.4.2. L-Aspartic acid b-tert-butyl ester a-[4-(2-
phenylethyl)]piperazinamide (35b, 35 with HNR1R2 = 4-
(phenylethyl)piperazine and n = 1). Use of N-benzyloxy-
carbonyl-L-aspartic acid b-tert-butyl ester a-[4-(2-phen-
ylethyl)]piperazinamide (5.89 g, 11.9 mmol) 34b gave
4.24 g (98%) of 35b as an off-white solid; 1H NMR
(CDCl3): d 1.42 (s, 9H); 2.61–2.95 (m, 10H); 3.60–3.90
(m, 4H); 4.35–4.45 (m, 1H); 7.17–7.29 (m, 5H).
4.1.4.8. D-Aspartic acid b-tert-butyl ester a-[N-
methyl-N-(3-trifluoromethylbenzyl)]amide (39e, 39 with
HNR1R2 = N-methyl-N-(3-trifluoromethylbenzyl)amine
and n = 1). Use of N-benzyloxycarbonyl-D-aspartic acid
b-tert-butyl ester a-[N-methyl-N-(3-trifluoromethylben-
zyl)]amide 38e (0.282 g, 0.57 mmol) gave 0.195 g (95%)
of 39e as an off-white solid. Compound 10e exhibited
4.1.4.3. L-Glutamic acid c-tert-butyl ester a-(3-trifluo-
romethyl)benzylamide (35c, 35 with HNR1R2 = 3-(tri-
fluoromethyl)benzylamine and n = 2). Use of N-
benzyloxycarbonyl-L-glutamic acid c-tert-butyl ester
a-(3-trifluoromethyl)benzylamide (5.41 g, 10.9 mmol)
34c gave 3.94 g (quantitative yield) of 35c as an off-
a
1H NMR spectrum consistent with the assigned
structure.
1
white solid; H NMR (CDCl3): d 1.41 (s, 9H); 1.73–
4.1.4.9. D-Aspartic acid b-tert-butyl ester a-[(S)-1-
1.89 (m, 3H); 2.05–2.16 (m, 1H); 2.32–2.38 (m, 2H);
3.47 (dd, J = 5.0 Hz, J = 7.5 Hz, 1H); 4.47–4.49 (m,
2H); 7.36–7.54 (m, 4H); 7.69–7.77 (m, 1H).
(3-trifluoromethylphenyl)ethyl]amide (39f, 39 with
HNR1R2 = (S)-1-(3-trifluoromethylphenyl)ethylamine and
n = 1). Use of N-benzyloxycarbonyl-D-aspartic acid
b-tert-butyl ester a-[(S)-1-(3-trifluoromethylphenyl)eth-
yl]amide 38f (120 mg, 0.24 mmol) gave 80 mg (91%) of
4.1.4.4. D-Aspartic acid b-tert-butyl ester a-(3-trifluo-
romethyl)benzylamide (39a, 39 with HNR1R2 = 3-(tri-
1
39f as an off-white solid; H NMR (MeOH-d4) d 1.42
fluoromethyl)benzylamine
and
n = 1).
Use
of
(s, 9H); 1.48 (d, J = 7.1 Hz, 3H); 2.58 (dd, J = 7.0 Hz,
J = 16.8 Hz, 1H); 2.69 (dd, J = 5.8 Hz, J = 16.8 Hz,
1H); 3.77–3.89 (m, 1H); 5.03–5.09 (m, 1H); 7.48–7.65
(m, 4H).
N-benzyloxycarbonyl-D-aspartic acid b-tert-butyl ester
a-(3-trifluoromethyl)benzylamide (1.41 g, 2.93 mmol)
38a gave 0.973 g (96%) of 39a as an off-white solid;
1H NMR (CDCl3): d 1.42 (s, 9H); 2.21 (br s, 2H);
2.67 (dd, J = 7.1 Hz, J = 16.8 Hz, 1H); 2.84 (dd,
J = 3.6 Hz, J = 16.7 Hz, 1H); 3.73–3.77 (m, 1H); 4.47–
4.50 (m, 2H); 7.41–7.52 (m, 4H); 7.83–7.87 (m, 1H).
4.1.4.10. D-Aspartic acid b-tert-butyl ester a-[(R)-
1-(3-trifluoromethylphenyl)ethyl]amide (39g, 39 with
HNR1R2 = (R)-1-(3-trifluoromethylphenyl)ethylamine and
n = 1). Use of N-benzyloxycarbonyl-D-aspartic acid
b-tert-butyl ester a-[(R)-1-(3-trifluoromethylphenyl)eth-
yl]amide 38g (217 mg, 0.44 mmol) gave 157 mg (quanti-
tative yield) of 39g as an off-white solid; 1H NMR
4.1.4.5. D-Aspartic acid b-tert-butyl ester a-(2-fluoro-3-
trifluoromethyl)benzylamide (39b, 39 with HNR1R2 = (2-
fluoro-3-trifluoromethyl)benzylamine and n = 1). Use of

C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
2067
(MeOH-d4) d 1.39 (s, 9H); 1.49 (d, J = 7.1 Hz, 3H); 2.55
(dd, J = 6.6 Hz, J = 16.4 Hz, 1H); 2.63 (dd, J = 5.7 Hz,
J = 16.4 Hz, 1H); 3.60–3.64 (m, 1H); 5.00–5.08 (m,
1H); 7.47–7.67 (m, 4H).
sulfate, filtered, and concentrated under reduced
pressure. The residue was either used directly for fur-
ther reactions, or purified by chromatography or by
crystallization from an appropriate solvent system
before use.
4.1.4.11. D-Glutamic acid c-tert-butyl ester a-(3-tri-
fluoromethyl)benzylamide (39h, 39 with HNR1R2 = 3-
(trifluoromethyl)benzylamine and n = 2). Use of N-ben-
zyloxycarbonyl-D-glutamic acid c-tert-butyl ester a-(3-
trifluoromethyl)benzylamide (1.667 g, 3.37 mmol) 38h
gave 1.15 g (94%) of 39h as an off-white solid; 1H
NMR (CDCl3) d 1.41 (s, 9H); 1.80–2.20 (m, 4H);
2.31–2.40 (m, 2H); 3.51–3.59 (m, 1H); 4.47–4.49 (m,
2H); 7.39–7.52 (m, 4H); 7.71–7.79 (m, 1H).
The following compounds were prepared according to
this sequence of procedures
4.1.5.3. tert-Butyl [3(S)-(4(S)-phenyloxazolidin-2-on-
3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetate (55). The
imine prepared from 4.53 g (34.5 mmol) of glycine tert-
butyl ester and t-cinnamaldehyde was combined with
2-(4(S)-phenyloxazolidin-2-on-3-yl) acetyl chloride (4)
to give 5.5 g (30%) of 55 as colorless crystals (recrystal-
lized, n-chlorobutane); mp 194–195 ꢁC. 1H NMR
(CDCl3) d 1.39 (s, 9H); 3.43 (d, J = 18.0 Hz, 1H); 4.15
(dd, J = 7.5 Hz, J = 8.7 Hz, 1H); 4.24 (d, J = 18.0 Hz,
1H); 4.55–4.63 (m, 3H); 4.80 (dd, J = 7.7 Hz,
J = 8.5 Hz, 1H); 6.05–6.13 (m, 1H); 6.64 (d,
J = 16.0 Hz, 1H); 7.28–7.42 (m, 10H).
4.1.4.12. L-Glutamic acid a-tert-butyl ester c-(4-
cyclohexyl)piperazinamide (44a, 44 with HNR1R2 = 1-
cyclohexylpiperazine and n = 2). Use of N-benzyloxycar-
bonyl-L-glutamic acid a-tert-butyl ester c-(4-cyclohex-
yl)piperazinamide (1.93 g, 3.96 mmol) 43a gave 1.30 g
1
(93%) of 44a as an off-white solid; H NMR (CDCl3)
d 1.02–1.25 (m, 5H); 1.41 (s, 9H); 1.45–1.50 (m, 1H);
1.56–1.60 (m, 1H); 1.69–1.80 (m, 6H); 3.30 (dd,
J = 4.8 Hz, J = 8.5 Hz, 1H); 3.44 (t, J = 9.9 Hz, 2H);
3.56 (t, J = 9.9 Hz, 2H).
4.1.5.4. 2(S)-(tert-Butoxycarbonylmethyl)-2-[3(S)-(4(S)-
phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-
1-yl]acetic acid N-(3-trifluoromethylbenzyl)amide (56).
Imine 36a prepared from 1.52 g (4.39 mmol) of L-aspartic
acid b-tert-butyl ester a-(3-trifluoromethyl)benzylamide
(35a) and t-cinnamaldehyde was combined with 2-(4(S)-
phenyloxazolidin-2-on-3-yl) acetyl chloride (4) to give
2.94 g of an orange-brown oil that gave, after flash col-
umn chromatography purification (70:30 hexanes/ethyl
4.1.5. General procedure for formation of a 2-azetidinone
from an imine and an acetyl chloride
4.1.5.1. Step 1. General procedure for formation of an
imine from an amino acid derivative. A solution of
1 equiv of an a-amino acid ester or amide (such as 35,
39 or 44) in dichloromethane was treated sequentially
with 1 equiv of an appropriate aldehyde (such as t-cin-
namaldehyde), and a desiccating agent, such as magne-
sium sulfate or silica gel, in the amount of about 2 g
of desiccating agent per gram of starting a-amino acid
ester or amide. The reaction mixture was stirred at
ambient temperature until all of the reactants were con-
sumed as measured by thin layer chromatography
(CH2Cl2 95%/MeOH 5%). When complete, the reaction
mixture was then filtered, the filter cake was washed
with dichloromethane, and the filtrate concentrated un-
der reduced pressure to provide the desired imine that
was used as it is in the subsequent step.
1
acetate), 2.06 g (70%) of 56 as a white solid; H NMR
(CDCl3)
d 1.39 (s, 9H); 2.46 (dd, J = 11.1 Hz,
J = 16.3 Hz, 1H); 4.18 (dd, J = 3.8 Hz, J = 16.4 Hz,
1H); 4.12–4.17 (m, 1H); 4.26 (d, J = 5.0 Hz, 1H); 4.45
(dd, J = 6.0 Hz, J = 14.9 Hz, 1H); 4.54 (dd, J = 5.3 Hz,
J = 9.8 Hz, 1H); 4.58–4.66 (m, 3H); 4.69–4.75 (m, 1H);
4.81 (dd, J = 3.8 Hz, J = 11.1 Hz, 1H); 6.25 (dd,
J = 9.6 Hz, J = 15.8 Hz, 1H); 6.70 (d, J = 15.8 Hz, 1H);
7.14–7.17 (m, 2H); 7.28–7.46 (m, 11H); 7.62 (s, 1H);
8.27–8.32 (m, 1H).
O
O
N
O
4.1.5.2. Step 2. General procedure for the [2 + 2]
cycloaddition of an imine and an acetyl chloride. A
dichloromethane solution of the imine (such as 36, 40,
or 45) (10 mL dichloromethane/1 g imine) was cooled
to 0 ꢁC. To this cooled solution was added 1.5 equiv
of an appropriate amine, typically triethylamine, fol-
lowed by the dropwise addition of a dichloromethane
solution of 1.1 equiv of an appropriate acetyl chloride
(such as 4) (10 mL of dichloromethane/1 g appropriate
acetyl chloride). The reaction mixture was allowed to
warm to ambient temperature over 1 h and was then
quenched by the addition of a saturated aqueous solu-
tion of ammonium chloride. The resulting mixture was
partitioned between water and dichloromethane. The
layers were separated and the organic layer was washed
successively with 1 N hydrochloric acid, saturated aque-
ous sodium bicarbonate, and saturated aqueous sodium
chloride. The organic layer was dried over magnesium
O
N
N
H
CF3
tBuO2C
4.1.5.5. 2(S)-(tert-Butoxycarbonylmethyl)-2-[3(S)-(4(S)-
phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-
1-yl]acetic acid N-[4-(2-phenylethyl)]piperazinamide (57).
Imine 36b prepared from 4.20 g (11.6 mmol) of L-
aspartic acid b-tert-butyl ester a-[4-(2-phenylethyl)]-
piperazinamide (35b) and t-cinnamaldehyde was
combined with 2-(4(S)-phenyloxazolidin-2-on-3-yl) ace-
tyl chloride (4) to give 4.37 g (55%) of 57 as a white
solid after flash column chromatography purification
(50:50 hexanes/ethyl acetate); 1H NMR (CDCl3) d
1.34 (s, 9H); 2.26–2.32 (m, 1H); 2.46–2.63 (m, 4H);

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C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
2.75–2.89 (m, 4H); 3.24–3.32 (m, 1H); 3.49–3.76 (m,
3H); 4.07–4.13 (m, 1H); 4.30 (d, J = 4.6 Hz, 1H);
4.22–4.48 (m, 1H); 4.55–4.61 (m, 1H); 4.69–4.75 (m,
1H); 5.04–5.09 (m, 1H); 6.15 (dd, J = 9.3 Hz,
J = 15.9 Hz, 1H); 6.63 (d, J = 15.8 Hz, 1H); 7.18–7.42
(m, 15H).
1H); 4.58–4.67 (m, 3H); 4.70–4.76 (m, 1H); 6.27 (dd,
J = 9.5 Hz, J = 15.8 Hz, 1H); 6.79 (d, J = 15.8 Hz,
1H); 7.25–7.50 (m, 13H); 7.63 (s, 1H); 8.50–8.54 (m,
1H).
O
O
N
O
O
O
O
N
N
O
N
O
H
CF3
N
tBuO2C
N
tBuO2C
N
4.1.5.8. 2(R)-(tert-Butoxycarbonylmethyl)-2-[3(S)-(4(S)-
phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-
yl]acetic acid N-(2-fluoro-3-trifluoromethylbenzyl)amide
(60). Imine 40b prepared from 0.256 g (0.70 mmol)
of D-aspartic acid b-tert-butyl ester a-(2-fluoro-3-tri-
fluoromethyl)benzylamide (39b) and t-cinnamaldehyde
was combined with 2-(4(S)-phenyloxazolidin-2-on-3-
yl) acetyl (4) to give 0.287 g (60%) of 60 as a white
solid after flash column chromatography purification
(70:30 hexanes/ethyl acetate); 1H NMR (CDCl3) d 1.38 (s,
9H); 3.12 (dd, J = 4.0 Hz, J = 17.8 Hz, 1H); 3.20 (dd,
J = 10.4 Hz, J = 17.8 Hz, 1H); 4.05 (dd, J = 3.9 Hz,
J = 10.4 Hz, 1H); 4.14 (dd, J = J0=8.2 Hz, 1H); 4.25 (d,
J = 4.9 Hz, 1H); 4.59–4.67 (m, 4H); 4.74 (t, J = 8.3 Hz,
1H); 6.36 (dd, J = 9.6 Hz, J = 15.8 Hz, 1H); 6.83 (d,
J = 15.8 Hz, 1H); 7.02–7.07 (m, 1H); 7.28–7.55 (m,
12H); 8.44–8.48 (m, 1H).
4.1.5.6. (S)-(tert-Butoxycarbonylethyl)-2-[3(S)-(4(S)-
phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-
1-yl]acetic acid N-(3-trifluoromethylbenzyl)amide (58).
Imine 36c prepared from 3.94 g (10.93 mmol) of
L-glutamic acid c-tert-butyl ester a-(3-trifluorometh-
yl)benzylamide (35c) and t-cinnamaldehyde was com-
bined with 2-(4(S)-phenyloxazolidin-2-on-3-yl) acetyl
chloride (4) to give 5.53 g (75%) of 58 as a white sol-
id after flash column chromatography purification
(70:30 hexanes/ethyl acetate); 1H NMR (CDCl3) d
1.36 (s, 9H); 1.85–1.96 (m, 1H); 2.18–2.49 (m, 3H);
4.14–4.19 (m, 1H); 4.30 (d, J = 4.9 Hz, 2H); 4.44
(dd, J = 6.1 Hz, J = 14.9 Hz, 1H); 4.56–4.67 (m,
4H); 4.71–4.75 (m, 1H); 6.26 (dd, J = 9.6 Hz,
J = 15.8 Hz, 1H); 6.71 (d, J = 15.8 Hz, 1H); 7.16–
7.18 (m, 2H); 7.27–7.49 (m, 11H); 7.60 (s, 1H);
8.08–8.12 (m, 1H).
O
O
N
O
O
O
O
N
F
N
O
N
H
O
CF3
tBuO2C
N
N
H
CF3
4.1.5.9. 2(R)-(tert-Butoxycarbonylmethyl)-2-[3(S)-(4(S)-
phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-
yl]acetic acid N-[(S)-a-methylbenzyl]amide (61). Imine
40c prepared from 0.167 g (0.57 mmol) of D-aspartic
acid b-tert-butyl ester [(S)-a-methylbenzyl]amide (39c)
and t-cinnamaldehyde was combined with 2-(4(S)-phe-
nyloxazolidin-2-on-3-yl) acetyl chloride (4) to give
0.219 g (63%) of 61 as a white solid after flash column
chromatography purification (70:30 hexanes/ethyl ace-
tate); 1H NMR (CDCl3) d 1.35 (s, 9H); 1.56 (d,
J = 7.0 Hz, 3H); 2.97 (dd, J = 3.5 Hz, J = 18.0 Hz, 1H);
3.15 (dd, J = 11.0 Hz, J = 17.5 Hz, 1H); 4.01 (dd,
J = 3.0 Hz, J = 11.0 Hz, 1H); 4.14 (t, J = 8.5 Hz, 1H);
4.24 (d, J = 5.0 Hz, 1H); 4.57 (dd, J = 5.0 Hz,
J = 9.5 Hz, 1H); 4.64 (t, J = 8.8 Hz, 1H); 5.07 (t,
J = 8.5 Hz, 1H); 5.03–5.09 (m, 1H); 6.43 (dd,
J = 9.5 Hz, J = 16.0 Hz, 1H); 6.83 (d, J = 16.0 Hz, 1H);
7.16–7.20 (m, 1H); 7.27–7.49 (m, 14H); 8.07–8.10 (m, 1H).
tBuO2C
4.1.5.7. 2(R)-(tert-Butoxycarbonylmethyl)-2-[3(S)-(4(S)-
phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-
yl]acetic acid N-(3-trifluoromethylbenzyl)amide (59).
Imine 40a prepared from 0.973 g (2.81 mmol) of D-as-
partic acid b-tert-butyl ester a-(3-trifluoromethyl)ben-
zylamide (39a) and t-cinnamaldehyde was combined
with 2-(4(S)-phenyloxazolidin-2-on-3-yl) acetyl chlo-
ride (4) to give 1.53 g (82%) of 59 as a white solid
after flash column chromatography purification
(70:30 hexanes/ethyl acetate); 1H NMR (CDCl3) d
1.37 (s, 9H); 3.10 (dd, J = 3.7 Hz, J = 17.8 Hz, 1H);
3.20 (dd, J = 10.7 Hz, J = 17.8 Hz, 1H); 4.02 (dd,
J = 3.6 Hz, J = 10.6 Hz, 1H); 4.11–4.17 (m, 1H); 4.24
(d, J = 4.9 Hz, 1H); 4.46 (dd, J = 5.8 Hz, J = 15.1 Hz,

C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
2069
J = 6.8 Hz, 1H); 4.70–4.79 (m, 2H); 5.27 (dd,
J = 4.0 Hz, J = 11.0 Hz, 1H); 6.22 (dd, J = 9.3 Hz,
J = 15.8 Hz, 1H); 6.73 (d, J = 15.8 Hz, 1H); 7.33–7.45
(m, 14H).
O
O
N
O
O
Me
N
N
H
O
O
tBuO2C
N
O
O
N
N
4.1.5.10. 2(R)-(tert-Butoxycarbonylmethyl)-2-[3(S)-
(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-
2-on-1-yl]acetic acid N-[(R)-a-methylbenzyl]amide (62).
Imine 40d prepared from 0.187 g (0.46 mmol) of D-as-
partic acid b-tert-butyl ester [(R)-a-methylbenzyl]amide
(39d) and t-cinnamaldehyde was combined with 2-
(4(S)-phenyloxazolidin-2-on-3-yl) acetyl chloride (4)
to give 0.25 g (64%) of 62 as a white solid after flash
column chromatography purification (70:30 hexanes/
ethyl acetate); 1H NMR (CDCl3) d 1.36 (s, 9H);
1.59 (d, J = 7.1 Hz, 3H); 3.10 (dd, J = 3.5 Hz,
J = 17.8 Hz, 1H); 3.22 (dd, J = 10.9 Hz, J = 17.8 Hz,
1H); 3.93 (dd, J = 3.5 Hz, J = 10.8 Hz, 1H); 4.14 (t,
J = 8.1 Hz, 1H); 4.24 (d, J = 5.0 Hz, 1H); 4.58 (dd,
J = 5.0 Hz, J = 9.5 Hz, 1H); 4.65 (t, J = 8.7 Hz, 1H);
4.74 (t, J = 8.2 Hz, 1H); 5.06–5.14 (m, 1H); 6.32 (dd,
J = 9.5 Hz, J = 15.8 Hz, 1H); 6.74 (d, J = 15.8 Hz,
1H); 7.19–7.43 (m, 15H); 8.15–8.18 (m, 1H). 13C
NMR (CDCl3) d 21.84, 27.98, 35.44, 49.56, 54.81,
61.00, 61.98, 63.60, 71.00, 81.15, 121.25, 126.33,
126.80, 126.87, 127.18, 128.41, 128.75, 128.78,
129.69, 135.27, 135.91, 139.03, 143.99, 157.97,
162.99, 167.26, 170.94.
CF3
tBuO2C
Me
4.1.5.12.
2(R)-(tert-Butoxycarbonylmethyl)-2-[3(S)-
(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-
on-1-yl]acetic acid N-[(S)-1-(3-trifluoromethylphenyl)eth-
yl]amide (64). Imine 40f prepared from 0.08 g
(0.22 mmol) of D-aspartic acid b-tert-butyl ester [(S)-
a-methylbenzyl]amide (39f) and t-cinnamaldehyde
was combined with 2-(4(S)-phenyloxazolidin-2-on-3-
yl) acetyl chloride (4) to give 53 mg (35%) of 64 as
a white solid after flash column chromatography puri-
fication (70:30 hexanes/ethyl acetate); 1H NMR
(CDCl3) d 1.36 (s, 9H); 1.59 (d, J = 7.0 Hz, 3H);
3.00 (dd, J = 3.4 Hz, J = 17.8 Hz, 1H); 3.19 (dd,
J = 11.0 Hz, J = 17.8 Hz, 1H); 4.01 (dd, J = 3.4 Hz,
J = 11.0 Hz, 1H); 4.07–4.19 (m, 2H); 4.25 (d,
J = 5.0 Hz, 1H); 4.58–4.78 (m, 3H); 5.09–5.16 (m,
1H); 6.44 (dd, J = 9.5 Hz, J = 15.8 Hz, 1H); 6.83 (d,
J = 15.8 Hz, 1H); 7.19–7.47 (m, 13H); 7.63 (s, 1H),
8.26–8.29 (m, 1H).
O
O
O
O
N
O
N
O
O
O
Me
Me
N
N
N
N
H
H
CF3
tBuO2C
tBuO2C
4.1.5.11. 2(R)-(tert-Butoxycarbonylmethyl)-2-[3(S)-
(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-
2-on-1-yl]acetic acid N-methyl-N-(3-trifluoromethylben-
zyl)amide (63). Imine 40e prepared from 0.195 g
(0.41 mmol) of D-aspartic acid b-tert-butyl ester a-[N-
methyl-N-(3-trifluoromethylbenzyl)]amide (39e) and t-
cinnamaldehyde was combined with 2-(4(S)-phenylox-
azolidin-2-on-3-yl) acetyl chloride (4) to give 0.253 g
(69%) of 63 as a white solid after flash column chroma-
4.1.5.13. 2(R)-(tert-Butoxycarbonylmethyl)-2-[3(S)-
(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-
2-on-1-yl]acetic acid N-[(R)-1-(3-trifluoromethylphe-
nyl)ethyl]amide (65). Imine 40g prepared from 0.16 g
(0.44 mmol) of D-aspartic acid b-tert-butyl ester [(R)-
a-methylbenzyl]amide (39g) and t-cinnamaldehyde
was combined with 2-(4(S)-phenyloxazolidin-2-on-3-
yl) acetyl chloride (4) to give 0.166 g (55%) of 65 as
a white solid after flash column chromatography puri-
fication (70:30 hexanes/ethyl acetate); 1H NMR
(CDCl3) d 1.36 (s, 9H); 1.60 (d, J = 7.1 Hz, 3H);
3.10 (dd, J = 3.7 Hz, J = 17.8 Hz, 1H); 3.19 (dd,
J = 10.6 Hz, J = 17.8 Hz, 1H); 3.93 (dd, J = 3.7 Hz,
J = 10.6 Hz, 1H); 4.16 (t, J = 8.1 Hz, 1H); 4.25 (d,
J = 5.0 Hz, 1H); 4.58 (dd, J = 5.0 Hz, J = 9.5 Hz,
1
tography purification (70:30 hexanes/ethyl acetate); H
NMR (CDCl3) d 1.36 (s, 9H); 2.53 (dd, J = 4.0 Hz,
J = 17.0 Hz, 1H); 3.06 (dd, J = 10.8 Hz, J = 16.8 Hz,
1H); 3.13 (s, 3H); 4.12 (dd, J = 8.0 Hz, J = 9.0 Hz,
1H); 4.26 (d, J = 5.0 Hz, 1H); 4.38 (d, J = 15.0 Hz,
1H); 4.46 (dd, J = 5.0 Hz, J = 9.5 Hz, 1H); 4.56 (t,

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C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
1H); 4.67 (t, J = 8.6 Hz, 1H); 4.74 (t, J = 8.2 Hz, 1H);
5.10–5.19 (m, 1H); 6.27 (dd, J = 9.5 Hz, J = 15.8 Hz,
1H); 6.74 (d, J = 15.8 Hz, 1H); 7.17–7.59 (m, 13H);
7.75 (s, 1H), 8.25–8.27 (m, 1H).
O
O
N
O
N
N
O
CO2tBu
O
O
N
O
N
O
Me
N
N
H
CF3
tBuO2C
4.1.6. General procedure for acylation of an (azetidin-2-
on-1-yl)acetate. A solution of (azetidin-2-on-1-yl)ace-
tate in tetrahydrofuran (0.22 M in azetidinone) is
cooled to ꢁ78 ꢁC and treated with lithium bis(trimeth-
ylsilyl)amide (2.2 equiv). The resulting anion is treated
with an appropriate acyl halide (1.1 equiv). Upon
complete conversion of the azetidinone, the reaction
is quenched with saturated aqueous ammonium chlo-
ride and partitioned between ethyl acetate and water.
The organic phase is washed sequentially with 1 N
hydrochloric acid, saturated aqueous sodium bicar-
bonate, and saturated aqueous sodium chloride. The
resulting organic layer is dried (magnesium sulfate)
and evaporated. The residue is purified by silica gel
chromatography with an appropriate eluent, such as
60:40 hexanes/ethyl acetate.
4.1.5.14. 2(R)-(tert-Butoxycarbonylethyl)-2-[3(S)-(4(S)-
phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-
1-yl]acetic acid N-(3-trifluoromethylbenzyl)amide (66).
Imine 40h prepared from 1.15 g (3.20 mmol) of D-glu-
tamic acid c-tert-butyl ester a-(3-trifluoromethyl)ben-
zylamide (39h) and t-cinnamaldehyde was combined
with 2-(4(S)-phenyloxazolidin-2-on-3-yl) acetyl chlo-
ride (4) to give 1.84 g (85%) of 66 as a white solid
after flash column chromatography purification
(70:30 hexanes/ethyl acetate); 1H NMR (CDCl3) d
1.37 (s, 9H); 2.23–2.39 (m, 4H); 3.71–3.75 (m, 1H);
4.13–4.18 (m, 1H); 4.31 (d, J = 4.9 Hz, 1H); 4.44–
4.51 (m, 2H); 4.56–4.68 (m, 2H); 4.71–4.76 (m, 1H);
6.26 (dd, J = 9.5 Hz, J = 15.8 Hz, 1H); 6.71 (d,
J = 15.8 Hz, 1H); 7.25–7.52 (m, 13H); 7.63 (s, 1H);
8.25–8.30 (m, 1H).
The following compound was prepared according to this
procedure:
4.1.6.1. 2,2,2-Trichloroethyl 2(R,S)-(tert-butoxycar-
bonyl)-2-[3(S)-(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-
(2-styryl)azetidin-2-on-1-yl]acetate (30). Azetidinone
55, 9.0 g (20 mmol), was acylated with 4.2 g (20 mmol)
of trichloroethylchloroformate to give 7.0 g (56%) of
30 as a white solid; mp 176–178 ꢁC. 1H NMR
(CDCl3) d 1.31–1.34 and 1.43–1.46 (m and m0, 9H);
4.12–4.18 (m, 1H); 4.53–4.90 (m; 6H); 5.18–5.24 (m,
1H); 6.10–6.18 (m, 1H); 6.61–6.66 (m, 1H); 7.28–7.41
(m, 10H).
O
O
N
O
O
N
N
H
CF3
tBuO2C
4.1.5.15. tert-Butyl 2(S)-(2-(4-cyclohexylpiperazin-1-
ylcarbonyl)ethyl)-2-[3(S)-(4(S)-phenyloxazolidin-2-on-3-
yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetate (67). Imine
45a prepared from 1.282 g (3.63 mmol) of L-glutamic
acid a-tert-butyl ester c-(4-cyclohexyl)piperazinamide
(44a) and t-cinnamaldehyde was combined with 2-
(4(S)-phenyloxazolidin-2-on-3-yl) acetyl chloride (4) to
give 1.946 g (80%) of 67 as a white solid after flash col-
umn chromatography purification (50:50 hexanes/ethyl
O
O
N
O
O
N
O
CCl3
tBuO2C
1
4.1.6.2.
2(RS)-(tert-Butoxycarbonyl)-2-[3(S)-(4(S)-
acetate); H NMR (CDCl3) d 1.15–1.26 (m, 6H); 1.39
phenyloxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-
yl]acetic acid N-(3-trifluoromethylbenzyl)amide (31). A
solution of 0.20 g (0.32 mmol) of 30 and 52 lL
(0.36 mmol) of (3-trifluoromethylbenzyl)amine in THF
was heated at reflux. Upon complete conversion
(TLC), the solvent was evaporated and the residue was
crystallized (chloroform/hexane) to give 0.17 g (82%)
(s, 9H); 1.55–1.64 (m, 2H); 1.77–1.83 (m, 3H); 2.22–
2.35 (m, 2H); 2.40–2.50 (m, 6H); 2.75–2.79 (m, 1H);
3.43–3.48 (m, 1H); 3.56–3.60 (m, 2H); 3.75–3.79 (m,
1H); 4.10 (t, J = 8.3 Hz, 1H); 4.31–4.35 (m, 2H); 4.58
(t, J = 8.8 Hz, 1H); 4.73 (t, J = 8.4 Hz, 1H); 6.17 (dd,
J = 8.6 Hz, J = 16.0 Hz, 1H); 6.65 (d, J = 16.0 Hz, 1H);
7.27–7.42 (m, 10H).

C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
2071
of 31 as a white solid; mp 182–184 ꢁC. 1H NMR
(CDCl3) d 1.36–1.44 (m, 9H); 4.13–4.14–4.91 (m; 8H);
6.23–6.31 (m, 1H); 6.65–6.78 (m, 1H); 7.20–7.60 (m,
14H); 7.88–7.92 and 8.08–8.12 (m and m0, 1H). Anal.
Calcd for C35H34F3N3O6: C, 64.71; H, 5.28; N, 6.47.
Found: C, 64.78; H, 5.31; N, 6.36.
O
O
N
O
O
N
N
H
CF3
HO2C
O
O
N
O
4.1.7.3. 2(S)-(Carboxymethyl)-2-[3(S)-(4(S)-phenylox-
azolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetic
acid N-[4-(2-phenylethyl)]piperazinamide (69). Com-
pound 57 (1.88 g, 2.78 mmol) was hydrolyzed to give
1.02 g (60%) of 69 as an off-white solid; 1H NMR
(CDCl3) d 2.63 (dd, J = 6.0 Hz, J = 16.5 Hz, 1H);
2.75–2.85 (m, 1H); 3.00 (dd, J = 8.2 Hz, J = 16.6 Hz,
1H); 3.13–3.26 (m, 4H); 3.37–3.56 (m, 4H); 3.86–4.00
(m, 1H); 4.05–4.11 (m, 1H); 4.24 (d, J = 5.0 Hz, 1H);
4.46–4.66 (m, 1H); 4.65–4.70 (m, 1H); 5.10–5.15 (m,
1H); 6.14 (dd, J = 9.3 Hz, J = 15.9 Hz, 1H); 6.71 (d,
J = 15.9 Hz, 1H); 7.22–7.41 (m, 15H); 12.02 (s, 1H).
O
N
N
H
tBuO2C
CF3
4.1.7. General procedure for hydrolysis of a tert-butyl
ester. A solution of tert-butyl ester derivative in formic
acid, typically 1 g in 10 mL, is stirred at ambient temper-
ature until no more ester is detected by thin-layer chro-
matography (dichloromethane 95%/methanol 5%), a
typical reaction time being around 3 h. The formic acid
is then evaporated under reduced pressure to yield the
corresponding carboxylic acid.
O
O
N
O
The following compounds were prepared according to
this procedure
O
N
N
4.1.7.1. 2(R,S)-(Carboxy)-2-[3(S)-(4(S)-phenyloxaz-
olidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetic
acid N-(3-trifluoromethylbenzyl)amide (32). Compound
31 (0.30 g, 0.46 mmol) was hydrolyzed to give 0.27 g
(quantitative yield) of 32 as an off-white solid; 1H
NMR (CDCl3) d 4.17–5.28 (m, 9H); 6.21–6.29 (m,
1H), 6.68–6.82 (m, 1H); 7.05–7.75 (m, 13H); 9.12–9.18
(m, 1H).
HO2C
N
4.1.7.4. 2(S)-(Carboxyethyl)-2-[3(S)-(4(S)-phenyloxazoli-
din-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetic acid
N-(3-trifluoromethylbenzyl)amide (70). Compound 58
(4.97 g, 7.34 mmol) was hydrolyzed to give 4.43 g
1
(97%) of 70 as an off-white solid; H NMR (CDCl3) d
1.92–2.03 (m, 1H); 2.37–2.51 (m, 3H); 4.13–4.19 (m,
1H); 3.32 (d, J = 4.9 Hz, 1H); 4.35–4.39 (m, 1H); 4.44
(dd, J = 5.9 Hz, J = 14.9 Hz, 1H); 4.50–4.57 (m, 2H);
4.61–4.67 (m, 1H); 4.70–4.76 (m, 1H); 6.24 (dd,
J = 9.6 Hz, J = 15.8 Hz, 1H); 6.70 (d, J = 15.8 Hz, 1H);
7.18–7.47 (m, 14H).
O
O
N
O
O
N
N
H
HO2C
CF3
O
O
N
O
4.1.7.2. 2(S)-(Carboxymethyl)-2-[3(S)-(4(S)-phenylox-
azolidin-2-on- 3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetic
acid N-(3-trifluoromethylbenzyl)amide (68). Compound
56 (1.72 g, 2.59 mmol) was hydrolyzed to give 1.57 g
(quantitative yield) of 68 as an off-white solid; 1H
NMR (CDCl3) d 2.61 (dd, J = 9.3 Hz, J = 16.6 Hz,
1H); 3.09–3.14 (m, 1H); 4.10–4.13 (m, 1H); 4.30 (d,
J = 4.5 Hz, 1H); 4.39–4.85 (m, 6H); 6.20 (dd,
J = 9.6 Hz, J = 15.7 Hz, 1H); 6.69 (d, J = 15.8 Hz, 1H);
7.12–7.15 (m, 2H); 7.26–7.50 (m, 11H); 7.61 (s, 1H);
8.41–8.45 (m, 1H).
O
N
N
H
CF3
HO2C
4.1.7.5. 2(R)-(Carboxymethyl)-2-[3(S)-(4(S)-phenylox-
azolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetic
acid N-(3-trifluoromethylbenzyl)amide (71). Compound

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C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
59 (1.51 g, 2.27 mmol) was hydrolyzed to give 1.38 g
(quantitative yield) of 71 as an off-white solid; 1H
NMR (CD3OD) d 2.93 (dd, J = 5.5 Hz, J = 17.0 Hz,
1H); 2.99 (dd, J = 9.0 Hz, J = 17.0 Hz, 1H); 4.17 (dd,
J = 7.0 Hz, J = 9.0 Hz, 1H); 4.36 (dd, J = 5.5 Hz,
J = 9.0 Hz, 1H); 4.40–4.45 (m, 2H) 4.55 (d,
J = 15.5 Hz, 1H); 4.65 (dd, J = 5.0 Hz, J = 9.0 Hz,
1H); 4.70 (dd, J = J0 = 9.0 Hz, 1H); 4.89 (dd,
J = 7.0 Hz, J = 9.0 Hz, 1H); 6.26 (dd, J = 9.0 Hz,
J = 16.0 Hz, 1H); 6.83 (d, J = 16.0 Hz, 1H); 7.24–7.32
(m, 5H); 7.38–7.48 (m, 6H); 7.51–7.57 (m, 2H); 7.64
(s, 1H). 13C NMR (CD3OD) d 43.82, 55.92, 62.01,
1H); 5.02–5.09 (m, 1H); 6.41 (dd, J = 9.4 Hz,
J = 15.8 Hz, 1H); 6.78 (d, J = 15.8 Hz, 1H); 7.18 (t,
J = 7.3 Hz, 1H); 7.26–7.43 (m, 14H); 8.29 (d,
J = 8.2 Hz, 1H).
O
O
N
O
O
Me
N
N
H
HO2C
3
63.34, 64.52, 72.58, 123.03, 124.97 (q, JC–F, 3.8Hz,
3
1C), 125.54 (q, JC–F, 3.8 Hz, 1C), 127.82, 128.55,
1
129.66, 129.81, 130.34, 130.44, 130.54, 131.77 (q, JC–
F, 32.3 Hz, CF3), 132.3–132.4 (m, 1C), 137.23, 138.42,
139.71, 141.19, 160.28, 166.12, 171.23.
4.1.7.8. 2(R)-(Carboxymethyl)-2-[3(S)-(4(S)-phenylox-
azolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]ace-
tic acid N-[(R)-a-methylbenzyl]amide (74). Compound 62
(0.22 g, 0.35 mmol) was hydrolyzed to give 0.20 g (quan-
titative yield) of 74 as an off-white solid; 1H NMR
(CDCl3) d 1.59 (d, J = 7.0 Hz, 1H); 3.25 (d, J = 7.0 Hz,
2H); 3.92 (t, J = 7.3 Hz, 1H); 4.15 (t, J = 8.3 Hz, 1H);
4.26 (d, J = 5.0 Hz, 1H); 4.52 (dd, J = 4.8 Hz,
J = 9.3 Hz, 1H); 4.65 (t, J = 8.8 Hz, 1H); 4.72 (t,
J = 8.3 Hz, 1H); 5.07–5.28 (m, 1H); 6.29 (dd,
J = 9.5 Hz, J = 15.6 Hz, 1H); 6.71 (d, J = 16.0 Hz, 1H);
7.20–7.43 (m, 15H); 8.31 (d, J = 8.0 Hz, 1H). 13C
NMR (CDCl3) d 21.73, 34.23, 49.83, 54.37, 61.02,
62.04, 63.64, 71.10, 120.71, 126.36, 126.91, 126.96,
127.20, 128.49, 128.78, 128.89, 129.74, 135.16, 135.84,
139.43, 143.68, 158.06, 163.37, 167.38, 174.53.
O
O
N
O
O
N
N
H
CF3
HO2C
4.1.7.6. 2(R)-(Carboxymethyl)-2-[3(S)-(4(S)-phenylox-
azolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetic
acid
N-(2-fluoro-3-trifluoromethylbenzyl)carboxamide
(72). Compound 60 (0.26 g, 0.38 mmol) was hydrolyzed
to give 0.238 g (quantitative yield) of 72 as an off-white
solid; 1H NMR (CDCl3) d 3.27 (d, J = 7.2 Hz, 1H); 4.06
(t, J = 7.2 Hz, 1H); 4.15 (t, J = 8.1 Hz, 1H); 4.27 (d,
J = 4.8 Hz, 1H); 4.56–4.76 (m, 5H); 6.34 (dd,
J = 9.5 Hz, J = 15.7 Hz, 1H); 6.80 (d, J = 15.7 Hz, 1H);
7.06 (t, J = 7.7 Hz, 1H); 7.31–7.54 (m, 12H); 8.58 (t,
J = 5.9 Hz, 1H).
O
O
N
O
O
Me
N
N
H
HO2C
4.1.7.9. 2(R)-(Carboxymethyl)-2-[3(S)-(4(S)-phenylox-
azolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]ace-
tic acid N-methyl-N-(3-trifluoromethylbenzyl)amide (75).
Compound 63 (0.253 g, 0.37 mmol) was hydrolyzed to
give 0.232 g (quantitative yield) of 75 as an off-white sol-
id; 1H NMR (CDCl3) d 3.07–3.15 (m, 4H); 4.13 (t,
J = 8.2 Hz, 1H); 4.30 (d, J = 4.9 Hz, 1H); 4.46–4.78
(m, 5H); 5.23 (dd, J = 4.6 Hz, J = 9.7 Hz, 1H); 6.20
(dd, J = 9.4 Hz, J = 15.9 Hz, 1H); 6.73 (d, J = 15.9 Hz,
1H); 7.25–7.43 (m, 14H).
O
O
N
O
O
N
F
N
H
CF3
HO2C
4.1.7.7. 2(R)-(Carboxymethyl)-2-[3(S)-(4(S)-phenylox-
azolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetic
acid N-[(S)-a-methylbenzyl]amide (73). Compound 61
(0.215 g, 0.35 mmol) was hydrolyzed to give 0.195 g
(quantitative yield) of 73 as an off-white solid; 1H
NMR (CDCl3) d 1.56 (d, J = 7.0 Hz, 1H); 3.10 (dd,
J = 4.5 Hz, J = 17.9 Hz, 1H); 3.18 (dd, J = 9.8 Hz,
J = 17.9 Hz, 1H); 4.00 (dd, J = 4.5 Hz, J = 9.7 Hz,
1H); 4.14 (t, J = 8.2 Hz, 1H); 4.26 (d, J = 4.7 Hz,
O
O
N
O
O
N
N
CF3
HO2C
Me

C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
2073
4.1.7.10. 2(R)-(Carboxylmethyl)-2-[3(S)-(4(S)-phenyl-
oxazolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-
yl]acetic acid N-[(S)-1-(3-trifluoromethylphenyl)ethyl]am-
ide (76). Compound 64 (38.5 mg, 0.06 mmol) was hydro-
lyzed to give 35 mg (quantitative yield) of 76 as an off-
O
O
N
O
O
N
1
white solid. H NMR (CDCl3) d 1.58 (d, J = 7.0 Hz,
N
H
3H); 3.07–3.25 (m, 2H); 4.02 (dd, J = 4.8 Hz,
J = 9.5 Hz, 1H); 4.15 (t, J = 8.2 Hz, 1H); 4.27 (d,
J = 4.8 Hz, 1H); 4.57 (dd, J = 4.8 Hz, J = 9.4 Hz, 1H);
4.61–4.77 (m, 2H); 5.07–5.29 (m, 1H); 6.41 (dd,
J = 9.4 Hz, J = 15.8 Hz, 1H); 6.79 (d, J = 15.8 Hz, 1H);
7.26–7.45 (m, 13H); 7.63 (s, 1H); 8.41 (d, J = 8.1 Hz,
1H).
CF3
HO2C
4.1.7.13. 2(S)-(2-(4-Cyclohexylpiperazin-1-ylcarbon-
yl)ethyl)-2-[3(S)-(4(S)- phenyloxazolidin-2-on-3-yl)-4(R)-
(2-styryl)azetidin-2-on-1-yl]acetic acid (79). Compound
67 (0.787 g, 1.28 mmol) was hydrolyzed to give 0.665 g
1
(92%) of 79 as an off-white solid; H NMR (CDCl3) d
1.05–1.13 (m, 1H); 1.20–1.40 (m, 5H); 1.60–1.64 (m,
1H); 1.79–1.83 (m, 2H); 2.00–2.05 (m, 2H); 2.22–2.44
(m, 3H); 2.67–2.71 (m, 1H); 2.93–3.01 (m, 4H); 3.14–
3.18 (m, 1H); 3.38–3.42 (m, 1H); 3.48–3.52 (m, 1H);
3.64–3.69 (m, 1H); 4.06–4.14 (m, 2H); 4.34–4.43 (m,
2H); 4.56 (t, J = 8.8 Hz, 1H); 4.73 (t, J = 8.4 Hz, 1H);
6.15 (dd, J = 9.1 Hz, J = 16.0 Hz, 1H); 6.65 (d,
J = 16.0 Hz, 1H); 7.25–7.42 (m, 10H).
O
O
N
O
O
Me
N
N
H
CF3
HO2C
4.1.7.11. 2(R)-(Carboxylmethyl)-2-[3(S)-(4(S)-phenyl-
oxazolidin-2-on- 3-yl)-4(R)-(2-styryl)azetidin-2-on-1-
O
O
N
O
N
yl]acetic acid N-[(R)-1-(3-trifluoromethylphenyl)ethyl]am-
ide (77). Compound 65 (166 mg, 0.24 mmol) was hydro-
lyzed to give 152 mg (quantitative yield) of 77 as an off-
N
O
CO2H
1
white solid. H NMR (CDCl3) d 1.60 (d, J = 7.0 Hz,
3H); 3.08–3.26 (m, 2H); 3.93 (dd, J = 4.7 Hz,
J = 9.5 Hz, 1H); 4.17 (t, J = 8.1 Hz, 1H); 4.26 (d,
J = 4.7 Hz, 1H); 4.54 (dd, J = 4.7 Hz, J = 9.4 Hz, 1H);
4.62–4.76 (m, 2H); 5.06–5.28 (m, 1H); 6.24 (dd,
J = 9.4 Hz, J = 15.8 Hz, 1H); 6.73 (d, J = 15.8 Hz, 1H);
7.28–7.58 (m, 13H); 7.74 (s, 1H); 8.40 (d, J = 7.4 Hz,
1H).
N
The compounds shown in Table 1, Figure 18 were pre-
pared using the procedure shown in Section 4.1.3, ex-
cept that N-benzyloxycarbonyl-D-aspartic acid b-tert-
butyl ester monohydrate was replaced with 32, and 3-
(trifluoromethyl)benzylamine was replaced with the
appropriate amine HNR1R2; all compounds exhibited
an 1H NMR spectrum consistent with the assigned
structure.
O
O
N
O
O
Me
N
N
H
CF3
HO2C
4.1.8. Aspartic acid derivatives. The compounds shown
in Table 2, Figure 19 were prepared using the procedure
shown in Section 4.1.3, except that N-benzyloxycarbon-
yl-D-aspartic acid b-tert-butyl ester monohydrate was
replaced with 68, and 3-(trifluoromethyl)benzylamine
was replaced with the appropriate amine HNR1R2; all
4.1.7.12. 2(R)-(Carboxyethyl)-2-[3(S)-(4(S)-phenylox-
azolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetic
acid N-(3-trifluoromethylbenzyl)amide (78). Compound
66 (0.604 g, 0.89 mmol) was hydrolyzed to give 0.554 g
(quantitative yield) of 78 as an off-white solid; 1H
NMR (CDCl3) d 2.27–2.34 (m, 4H); 4.10–4.30 (m,
2H); 4.30 (d, J = 4.3 Hz, 1H); 4.40–4.64 (m, 4H); 4.47
(t, J = 8.1 Hz, 1H); 6.22 (dd, J = 9.4 Hz, J = 15.7 Hz,
1H); 6.70 (d, J = 15.7 Hz, 1H); 7.25–7.57 (m, 14H);
8.32–8.36 (m, 1H).
1
compounds exhibited an H NMR spectrum consistent
with the assigned structure.
The compounds shown in Table 3, Figure 20 were
prepared using the procedure shown in Section 4.1.3,
except that N-benzyloxycarbonyl-D-aspartic acid b-
tert-butyl ester monohydrate was replaced with 69,
and 3-(trifluoromethyl)benzylamine was replaced with

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C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
the appropriate amine HNR1R2; all compounds exhibit-
ed an H NMR spectrum consistent with the assigned
structure.
1
O
O
N
N
O
O
The compounds shown in Table 4, Figure 21 were pre-
pared using the procedure shown in Section 4.1.3, except
that N-benzyloxycarbonyl-D-aspartic acid b-tert-butyl
ester monohydrate was replaced with 71, and 3-(trifluo-
romethyl)benzylamine was replaced with the appropri-
ate amine HNR1R2; all compounds exhibited an 1H
NMR spectrum consistent with the assigned structure
(Table 5, HRMS and Ki data).
N
N
H
F
O
N
F3C
4.1.9.2. 2(R)-[[4-(Piperidin-1-yl)piperidin-1-yl]carbon-
ylmethyl]-2-[3(S)-(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-
(2-styryl)azetidin-2-on-1-yl]acetic acid N-[(S)-a-methylb-
enzyl]amide (52). Compound 52 was prepared using
the procedure shown in Section 4.1.3, except that N-ben-
zyloxycarbonyl-D-aspartic acid b-tert-butyl ester
monohydrate was replaced with 73 and 3-(trifluoro-
methyl)benzylamine was replaced with 4-(1-piperidi-
nyl)piperidine. Compound 52 exhibited a 1H NMR
spectrum consistent with the assigned structure. HRMS
(FAB) calcd for C42H50N5O5 704.3812, found 704.3806
(M+H)+.
4.1.9. A typical example being 41: 2(R)-[[4-(piperidin-1-
yl)piperidin-1-yl]carbonylmethyl]-2-[3(S)-(4(S)-phenylox-
azolidin-2-on-3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]ace-
tic acid N-(3-trifluoromethylbenzyl)amide. Compound 41
was obtained as an off-white solid after flash silica gel
column chromatography (CH2Cl2 99% and down to
90%/MeOH 1% and up to 10%, NH4OH < 1%). 1H
NMR (CDCl3) d 1.34–1.54 (m, 4H); 1.60–1.74 (m,
4H); 1.84–1.95 (m, 2H); 2.42–2.66 (m, 6H); 2.87–2.95
(m, 1H); 3.13–3.19 (m, 1H); 3.30–3.37 (m, 1H); 3.77–
3.84 (m, 1H); 4.09–4.18 (m, 2H); 4.22 (dd, J = 4.5 Hz,
1H); 4.43–4.56 (m, 2H); 4.58–4.66 (m, 2H); 4.70–4.74
(m, 2H); 6.25–6.32 (m, 1H); 6.80–6.85 (m, 1H); 7.25–
7.53 (m, 13H); 7.64 (s, 1H); 8.62–8.69 (m, 1H). 13C
NMR (CDCl3) d 24.02, 25.30, 27.10, 29.63, 33.72/
33.75, 41.18/41.37, 43.15, 44.56/44.67, 49.90/49.98,
54.79/54.92, 61.16, 61.94/61.98, 62.50/62.62, 63.30/
O
O
N
O
3
63.34, 71.06, 120.69, 123.05, 123.77 (q, JC–F, 3.5 Hz,
1C), 124.52 (q, JC–F, 3.8 Hz, 1C), 125.21, 126.78,
O
Me
3
N
N
127.27, 128.77, 128.85, 128.92, 129.65, 129.74, 130.55,
130.76/130.79, 135.27, 135.68/135.72, 139.43, 139.48/
139.53, 158.30, 163.18, 168.07/168.34, 169.41. HRMS
(FAB) calcd for C42H47F3N5O5 758.3529, found
758.3510 (M+H)+.
H
N
O
N
O
O
4.1.9.3. 2(R)-[[ 4-[2-(1-Piperidinyl)ethyl]piperidin-1-
yl]carbonylmethyl]-2-[3(S)-(4(S)-phenyloxazolidin-2-on-
3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetic acid N-[(S)-
a-methylbenzyl]amide (103). Compound 103 was pre-
pared using the procedure shown in Section 4.1.3, except
that N-benzyloxycarbonyl-D-aspartic acid b-tert-butyl
ester monohydrate was replaced with 73 and 3-(trifluo-
romethyl)benzylamine was replaced with 4-[2-(1-pipe-
N
O
O
N
N
H
N
O
N
F3C
1
ridinyl)ethyl]piperidine. Compound 103 exhibited a H
NMR spectrum consistent with the assigned structure.
HRMS (FAB) calcd for C44H54N5O5 732.4125,
732.4108 (M+H)+.
4.1.9.1. 2(R)-[[4-(Piperidin-1-yl)piperidin-1-yl]carbon-
ylmethyl]-2-[3(S)-(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-
(2-styryl)azetidin-2-on-1-yl]acetic acid N-(2-fluoro-3-tri-
fluoromethylbenzyl)amide (102). Compound 102 was
prepared using the procedure shown in Section 4.1.3, ex-
cept that N-benzyloxycarbonyl-D-aspartic acid b-tert-
butyl ester monohydrate was replaced with 72 and 3-(tri-
fluoromethyl)benzylamine was replaced with 4-(1-pipe-
ridinyl)piperidine. Compound 102 exhibited a 1H
NMR spectrum consistent with the assigned structure.
HRMS (FAB) calcd for C42H46F4N5O5 776.3435, found
776.3463 (M+H)+.
O
O
N
N
O
O
Me
N
N
H
O
N

C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
Table 1. Structures, HRMS (FAB), V1a Ki values
2075
Compound
HNR1R2
Calcd for and equal to
Found: (M+H)
Ki (nM)
33
80
81
82
83
84
85
4-[2-(1-Piperidinyl)ethyl]piperidine
4-(1-Piperidinyl)piperidine
4-Butylpiperazine
C
43H49F3N5O5 772.3686
772.3711
744.3399
718.3194
766.3191
704.3038
744.3367
704.3064
1.17
3.39
6.5
C41H45F3N5O5 744.3373
C39H43F3N5O5 718.3216
C43H43F3N5O5 766.3216
1-Benzyl-4-aminopiperidine
4-Isopropylpiperazine
13.3
C
C
38H41F3N5O5 704.3060
41H45F3N5O5 744.3377
18.2
26.4
61
4-Cyclohexylpiperazine
2-(1-Piperidinyl)ethylamine
C38H41F3N5O5 704.3060
O
O
O
O
HNR1R2
HOAT
EDC, HCl
CH2Cl2
N
O
N
O
O
O
N
N
OH
NR1R2
HN
HN
F3C
F3C
O
O
32
Figure 18. Aminomalonic acid derivatives.
Table 2. Structures, HRMS (FAB), V1a Ki values
Compound
HNR1R2
Calcd for and equal to
Found: (M+H)
Ki (nM)
37
42
86
87
88
89
90
91
92
93
1-Benzyl-4-aminopiperidine
4-(1-Piperidinyl)piperidine
C
44H45F3N5O5 780.3373
42H47F3N5O5 758.3529
780.3356
758.3554
766.3201
794.3511
786.3860
782.3535
744.3355
758.3555
744.3397
773.3638
1.13
1.89
3.60
5.12
6.00
23
C
(+)-3(S)-1-Benzyl-3-aminopyrrolidine
4-Benzylhomopiperazine
4-[2-(1-Piperidinyl)ethyl]piperidine
1-Benzyl-4-aminopiperidinea (Zone B = 2-phenylethyl)
C43H43F3N5O5 766.3216
45H47F3N5O5 794.3524
C
C44H51F3N5O5 786.3842
C44H47F3N5O5 782.3534
C41H45F3N5O5 744.3373
4-(1-Pyrrolidinyl)piperazine
4-Cyclohexylpiperazine
ꢀ30
ꢀ30
<60
<60
C
42H47F3N5O5 758.3529
C41H45F3N5O5 744.3373
C42H48F3N6O5 773.3638
4-Cyclopentylpiperazine
4-[2-(1-Pyrrolidinyl)ethyl]piperazine
a A suspension of 0.05 g (0.064 mmol) of 37 and palladium (5% weight on activated carbon, 0.024 g) in 5 mL of methanol was held under an
atmosphere of hydrogen until complete disappearance of 37 as determined by thin layer chromatography (95:5 dichloromethane/methanol eluent).
The reaction mixture was filtered to remove the palladium over carbon and the filtrate was evaporated to give 0.04 g of crude 89. It was purified by
column chromatography (95:5 dichloromethane/methanol eluent) to afford 0.011 g (23%) of 89. HRMS (FAB) calcd for C44H47F3N5O5 782.3534,
found 782.3535 (M+H)+.
O
O
O
O
HNR1R2
HOAT
EDC, HCl
CH2Cl2
N
O
N
O
N
N
OH
NR1R2
H
N
H
N
F3C
F3C
O
O
O
O
68
Figure 19. L-Aspartic acid derivatives.
4.1.9.4. 2(R)-[[4-(Piperidin-1-yl)piperidin-1-yl]carbon-
ylmethyl]-2-[3(S)-(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-
(2-styryl)azetidin-2-on-1-yl]acetic acid N-[(R)-a-methylb-
enzyl]amide (50). Compound 50 was prepared using
the procedure shown in Section 4.1.3, except that N-ben-
zyloxycarbonyl-D-aspartic acid b-tert-butyl ester
monohydrate was replaced with 74 and 3-(trifluoro-
methyl)benzylamine was replaced with 4-(1-piperidi-
nyl)piperidine. Compound 50 was obtained as an
off-white solid after flash silica gel column chromatogra-
phy (CH2Cl2 99% and down to 90%/MeOH 1% and up
1
to 10%, NH4OH <1%). H NMR (CDCl3) d 1.30–1.52

2076
C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
Table 3. Structures, HRMS (FAB), V1a Ki values
Compound
HNR1R2
Calcd for and equal to
44H45F3N5O5 780.3373
Found: (M+H)
Ki (nM)
94
95
96
3-(Trifluoromethyl)benzylamine
2-(Dimethylamino)ethylamine
3-(Dimethylamino)propylamine
C
780.3344
693.3757
707.3912
6100
>600
>600
C40H49F3N6O5 693.3764
C41H51N6O5 707.3921
O
N
O
N
HNR1R2
HOAT
EDC, HCl
CH2Cl2
O
N
O
N
O
N
O
N
N
OH
NR1R2
N
O
O
O
O
69
Figure 20. D-Aspartic acid derivatives.
Table 4. Structures and HRMS (FAB), V1a Ki values
Compound
HNR1R2
Calcd for and equal to
Found: (M+H)
Ki (nM)
41
97
4-(1-Piperidinyl)piperidine
4-[(1-Piperidinyl)methyl]piperidine
1-Benzyl-4-amino-piperidine
C
42H47F3N5O5 758.3529
43H49F3N5O5 772.3686
758.3510
772.3666
780.3353
786.3840
744.3398
766.3223
1.49
3.7
C
98
99
C44H45F3N5O5 780.3373
C44H51F3N5O5 786.3842
C41H45F3N5O5 744.3373
5.58
7.9
4-[2-(1-Piperidinyl)ethyl]piperidine
4-(1-Pyrrolidinyl)piperazine
3-(S)-(1-Benzyl)-3-amino-pyrrolidine
100
101
27.5
>30
C43H43F3N5O5 766.3216
HNR1R2
HOAT
EDC, HCl
CH2Cl2
O
O
O
O
N
O
N
O
N
N
OH
NR1R2
O
H
N
H
F3C
F3C
N
O
O
O
71
Figure 21. Additional D-aspartic acid derivatives.
Table 5. HRMS (FAB), V1a Ki values
Compound
Calcd for and equal to
42H50N5O5 704.3812
Found: (M+H)
Ki (nM)
50
51
C
704.3801
772.3674
776.3463
772.3703
732.4127
772.3666
704.3806
732.4108
0.30
0.66
1.13
1.27
8.7
C43H49F3N5O5 772.3686
C42H46F4N5O5 776.3435
102
53
C
43H49F3N5O5 772.3686
C44H54N5O5 732.4125
43H49F3N5O5 772.3686
C42H50N5O5 704.3812
44H54N5O5 732.4125
104
105
52
C
>6
>30
179
103
C
(m, 4H); 1.56–1.72 (m, 7H); 1.81–1.90 (m, 2H); 2.40–
2.60 (m, 6H); 2.88–2.96 (m, 1H); 3.14–3.20 (m, 1H);
3.36–3.44 (m, 1H); 3.79–3.86 (m, 1H); 3.99–4.04 (m,
1H); 4.15 (t, J = 8.3 Hz, 1H); 4.21 (dd, J = 5.0 Hz,
J = 8.0 Hz, 1H); 4.44–4.55 (m, 1H); 4.60–4.75 (m,
3H); 5.07–5.14 (m, 1H); 6.29–6.36 (m, 1H); 6.74–

C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
2077
6.79 (m, 1H); 7.19–7.46 (m, 15H); 8.23–8.28 (m, 1H).
13C NMR (CDCl3) d 21.87/21.90, 23.84, 24.90, 26.81,
33.56/33.59, 41.11/41.23, 44.52/44.57, 49.59, 49.67/
49.75, 54.97, 55.16, 61.03, 61.77/61.81, 62.43/62.52,
63.37, 70.95, 121.16, 126.30, 126.79, 126.83, 127.25,
128.40, 128.70, 129.63, 129.67, 135.29/135.31,
tent with the assigned structure. HRMS (FAB) calcd
for C43H49F3N5O5 772.3686, found 772.3674
(M+H)+.
O
O
135.80/135.83,
139.13,
143.93/143.96,
157.96,
N
O
163.29, 167.94/167.96, 168.23, 168.51. HRMS (FAB)
calcd for C42H50N5O5 704.3812, found 704.3801
(M+H)+.
O
N
N
Me
N
O
O
O
N
N
F3C
N
O
O
Me
N
N
H
4.1.9.7. 2(R)-[[4-(Piperidin-1-yl)piperidin-1-yl]carbon-
ylmethyl]-2-[3(S)-(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-
(2-styryl)azetidin-2-on-1-yl]acetic acid N-[(R)-a-methyl-
3-trifluoromethylbenzyl]amide (53). Compound 53 was
prepared using the procedure shown in Section 4.1.3,
except that N-benzyloxycarbonyl-D-aspartic acid b-
tert-butyl ester monohydrate was replaced with 77 and
3-(trifluoromethyl)benzylamine was replaced with 4-(1-
piperidinyl)piperidine. Compound 53 was obtained as
an off-white solid after flash silica gel column chroma-
tography (CH2Cl2 99% and down to 90%/MeOH 1%
and up to 10%, NH4OH < 1%). Compound 53 exhibited
a 1H NMR spectrum consistent with the assigned struc-
ture. HRMS (FAB) calcd for C43H49F3N5O5 772.3686,
found 772.3703 (M+H)+.
O
N
4.1.9.5. 2(R)-[[ 4-[2-(1-Piperidinyl)ethyl]piperidin-1-
yl]carbonylmethyl]-2-[3(S)-(4(S)-phenyloxazolidin-2-on-
3-yl)-4(R)-(2-styryl)azetidin-2-on-1-yl]acetic acid N-[(R)-
a-methylbenzyl]amide (104). Compound 104 was
prepared using the procedure shown in Section 4.1.3’,
except that N-benzyloxycarbonyl-D-aspartic acid
b-tert-butyl ester monohydrate was replaced with 74
and 3-(trifluoromethyl)benzylamine was replaced with
4-[2-(1-piperidinyl)ethyl]piperidine. Compound 104
1
exhibited a H NMR spectrum consistent with the as-
signed structure. HRMS (FAB) calcd for C44H54N5O5
732.4125, 732.4127 (M+H)+.
O
O
N
O
O
O
N
O
Me
N
N
O
N
H
O
Me
N
N
O
N
H
N
F3C
O
N
4.1.9.8. 2(R)-[[4-(Piperidin-1-yl)piperidin-1-yl]carbon-
ylmethyl]-2-[3(S)-(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-(2-
styryl)azetidin-2-on-1-yl]acetic acid N-[(S)-a-methyl-3-
trifluoromethylbenzyl]amide (105). Compound 105 was
prepared using the procedure shown in Section 4.1.3,
except that N-benzyloxycarbonyl-D-aspartic acid b-
tert-butyl ester monohydrate was replaced with 76 and
3-(trifluoromethyl)benzylamine was replaced with 4-(1-
piperidinyl)piperidine. Compound 105 was obtained as
an off-white solid after flash silica gel column chroma-
tography (CH2Cl2 99% and down to 90%/MeOH 1%
and up to 10%, NH4OH <1%). Compound 105 exhibit-
4.1.9.6. 2(R)-[[4-(Piperidin-1-yl)piperidin-1-yl]carbon-
ylmethyl]-2-[3(S)-(4(S)-phenyloxazolidin-2-on-3-yl)-4(R)-
(2-styryl)azetidin-2-on-1-yl]acetic acid N-methyl-N-(3-
trifluoromethylbenzyl)amide (51). Compound 51 was
prepared using the procedure shown in Section
4.1.3, except that N-benzyloxycarbonyl-D-aspartic acid
b-tert-butyl ester monohydrate was replaced with 75
and 3-(trifluoromethyl)benzylamine was replaced with
4-(1-piperidinyl)piperidine. Compound 51 was ob-
tained as an off-white solid after flash silica gel col-
umn chromatography (CH2Cl2 99% and down to
90%/MeOH 1% and up to 10%, NH4OH <1%).
1
ed a H NMR spectrum consistent with the assigned
structure. HRMS (FAB) calcd for C43H49F3N5O5
1
Compound 51 exhibited a H NMR spectrum consis-
772.3686, found 772.3666 (M+H)+.

2078
C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
serum and 250 lg/ml G418 (Gibco, Grand Island,
O
O
N
NY, USA). For competitive binding assay, hV1a cells
were plated into 12-well culture plate at 1:16 dilution
from a confluency flask and maintained in culture for
at least two days. Culture medium was then removed,
cells were washed with 2 mL binding buffer (25 mM
HEPES, 0.25% BSA, 1· DMEM, pH 7.0). To each
well, 990 ll binding buffer containing 1 nM [3H]argi-
nine–vasopressin was added, and followed by 10 ll
series diluted test compounds dissolved in DMSO.
All incubations were in triplicate, and dose-inhibition
curves consisted of total binding (DMSO) and 5 con-
centrations (usually 0.1, 1.0, 10, 100, and 1000 nm) of
test agent encompassing the IC50. 100 nM unlabeled
arginine–vasopressin (Sigma) was used to assess non-
specific binding. Cells were incubated for 30 min at
37 ꢁC, assay mixture was removed, and each well
was washed three times with PBS (pH 7.4). SDS
(1 mL, 2%) was added per well and plates were let
sit for 30 min. The whole content in a well was trans-
ferred to a scintillation vial. Each well was rinsed with
0.5 mL PBS, which was then added to the correspond-
ing vial. Scintillation fluid (Ecoscint, National Diag-
nostics, Atlanta, Georgia) was then added at 3 mL
per vial. Samples were counted in a liquid scintillation
counter (Beckman LS6500). IC50 values were calculat-
ed by Prism Curve fitting software. The conversion
factor for computing Ki from IC50 was 0.61.
N
O
O
Me
N
N
H
O
N
F3C
4.1.10. Glutamic acid derivatives. The compounds shown
in Table 6, Figure 22 were prepared using the procedure
shown in Section 4.1.3, except that N-benzyloxycarbon-
yl-D-aspartic acid b-tert-butyl ester monohydrate was
replaced with 70, and 3-(trifluoromethyl)benzylamine
was replaced with the appropriate amine HNR1R2; all
1
compounds exhibited an H NMR spectrum consistent
with the assigned structure.
The compounds shown in Table 7, Figure 23 were pre-
pared using the procedure shown in Section 4.1.3, except
that N-benzyloxycarbonyl-D-aspartic acid b-tert-butyl
ester monohydrate was replaced with 79, and 3-(trifluo-
romethyl)benzylamine was replaced with the appropri-
ate amine HNR1R2; all compounds exhibited an 1H
NMR spectrum consistent with the assigned structure.
4.2.2. V1a receptor binding assay (Eli Lilly & Co). The
V1a binding assay employed at Eli Lilly used the same
cell line expressing the human V1a receptor in CHO
cells, but the assay differed inasmuch as binding was car-
ried out on membranes using a filtration method.22 IC50
values measured by the two methods usually differed by
2-fold or less.
The compounds shown in Table 8, Figure 24
were prepared using the procedure shown in Section
4.1.3, except that N-benzyloxycarbonyl-D-aspartic acid
b-tert-butyl ester monohydrate was replaced with 78,
and 3-(trifluoromethyl)benzylamine was replaced
with the appropriate amine HNR1R2; all compounds
exhibited an 1H NMR spectrum consistent with the
assigned structure.
4.2.3. Measurement of plasma and brain levels in dogs
after oral dosing. Samples were collected from a single
dog dosed orally with compound at 10 mg/kg in 10%
DMSO/90% PEG300. Blood and brain samples (cor-
tex, brain stem, cerebellum, hypothalamus, and hip-
pocampus regions) were harvested and frozen in
liquid nitrogen and stored at ꢁ70 ꢁC prior to analysis
by LC/MS mass spectrometry. Samples were thawed
on ice, homogenized in pH 9.4 buffer on ice and
extracted with ethyl acetate after addition of a refer-
ence compound to measure extraction efficiency.
Extracts were removed and taken to dryness, reconsti-
tuted in acetonitrile and injected on a Finnigan ESI
LC/MS/MS system. Spiked control brain samples
4.2. Biological evaluation
4.2.1. V1a receptor binding assay (Azevan Pharmaceuti-
cals Inc.). A cell line expressing the human V1a recep-
tor in CHO cells (henceforth referred to as the hV1a
cell line) was obtained from Dr. Michael Brownstein,
NIMH, Bethesda, MD, USA. The hV1a cell culture
and cell-based hV1a receptor binding assay were per-
formed according to the methods described by Thi-
bonnier et al.26 with modifications. The hV1a cell
line was grown in alpha-MEM with 10% fetal bovine
Table 6. Structures, HRMS (FAB), V1a IC50 and Ki values
Compound
HNR1R2
Calcd for and equal to
43H49F3N5O5 772.3686
C44H51F3N5O5 786.3842
45H47F3N5O5 794.3529
C43H49F3N5O5 772.3686
40H45F3N5O5 732.3373
C44H51F3N5O5 786.3842
40H45F3N5O5 732.3373
C45H53F3N5O5 800.3999
Found: (M+H)
Ki (nM)
48
106
46
4-Cyclohexylpiperazine
C
772.3672
786.3862
794.3502
772.3685
732.3371
786.3821
732.3374
800.4019
0.27
0.46
1.17
1.45
1.19
1.95
9
4-(Cyclohexylmethyl)piperazine
4-(2-Phenylethyl)piperazine
4-(1-Piperidinyl)piperidine
4-Propylpiperazine
C
47
107
108
109
110
C
4-[(1-Piperidinyl)methyl]piperidine
2-(1-Piperidinyl)ethylamine
4-[2-(1-Piperidinyl)ethyl]piperidine
C
9.9

C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
2079
O
O
HNR1R2
O
O
HOAT
EDC, HCl
CH2Cl2
N
O
N
O
O
O
N
N
OH
NR1R2
H
N
H
N
F3C
F3C
O
O
70
Figure 22. L-Glutamic acid derivatives.
Table 7. Structures, HRMS (FAB), V1a Ki values
Compound
HNR1R2
Calcd for and equal to
43H48F4N5O5 790.3592
C42H48Cl2N5O5 772.3033
43H48F4N5O5 790.3592
C43H48F4N5O5 790.3592
42H49ClN5O5 738.3422
C42H48F2N5O5 740.3624
43H49F3N5O5 772.3686
Found (M+H)
Ki (nM)
111
112
113
114
115
116
117
118
119
2-Fluoro-3-(trifluoromethyl)benzylamine
2,3-Dichlorobenzylamine
C
790.3572
772.3011
790.3568
790.3577
738.3441
740.3624
772.3692
840.3583
790.3588
0.20
0.38
0.41
0.51
0.66
0.82
0.93
4.45
2-Fluoro-5-(trifluoromethyl)benzylamine
3-Fluoro-5-(trifluoromethyl)benzylamine
3-Chlorobenzylamine
C
C
3,5-Difluorobenzylamine
2-(Trifluoromethyl)benzylamine
3,5-Bis-(trifluoromethyl)benzylamine
4-Fluoro-3-(trifluoromethyl)benzylamine
C
C44H48F6N5O5 840.3560
C43H48F4N5O5 790.3592
<30
O
O
O
O
HNR1R2
HOAT
EDC, HCl
CH2Cl2
N
O
N
O
O
O
N
N
N
N
O
O
OH
NR1R2
N
N
79
Figure 23. Additional L-glutamic acid derivatives.
Table 8. Structures, HRMS (FAB), V1a Ki values
Compound
HNR1R2
Calcd for and equal to
43H49F3N5O5 772.3686
C41H47F3N5O5 746.3529
Found (M+H)
Ki (nM)
49
120
121
122
123
124
4-Cyclohexylpiperazine
4-Butylpiperazine
4-Isopropylpiperazine
C
772.3707
746.3503
732.3347
794.3552
786.3823
772.3688
5.84
10.5
13
C
40H45F3N5O5 732.3373
45H47F3N5O5 794.3529
44H51F3N5O5 786.3842
4-(2-Phenylethyl)piperazine
4-(Cyclohexylmethyl)-piperazine
4-(1-Piperidinyl)piperidine
C
C
33
>12
ꢀ30
C43H49F3N5O5 772.3686
O
O
O
O
HNR1R2
HOAT
EDC, HCl
CH2Cl2
N
O
N
O
OH
NR1R2
O
N
N
O
HN
HN
F3C
F3C
O
O
78
Figure 24. D-Glutamic acid derivatives.

2080
C. D. Guillon et al. / Bioorg. Med. Chem. 15 (2007) 2054–2080
11. Xiang, M. A.; Chen, R. H.; Demarest, K. T.; Gunnet, J.;
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were prepared in similar fashion for use as quantita-
tion standards.
4.2.4. Measurement of plasma and brain levels in rats
after oral dosing. Initial experiments showed that in
most cases our compounds could be dissolved at
20 mg/ml in 23% hydroxypropyl b-cyclodextrin with
0.68% lactic acid. Sprague–Dawley rats (Charles River
Laboratory) were dosed by gavage with a combination
of three compounds (20 mg/kg each): 22 + 41 + 48 or
42 + 46 + 47. Blood (approximately 3 mL) was collected
from three animals per group per time point at 0.25, 0.5,
1, 2, 4, 8, 12, and 24 h post-dose. Blood was collected via
exsanguination (cardiac puncture) under carbon dioxide
anesthesia into tubes containing sodium heparin antico-
agulant and plasma was separated by centrifugation
within 1 h. Brains were collected at the same time points.
Plasma and brain levels were determined by LC/MS/
MS.
16. This argument is very similar to the concept underlying
‘privileged fragments’ Evans, B. E.; Rittle, K. E.; Bock, M.
F. G.; Dipardo, R. M.; Friedinger, R. M.; Whiter, W. L.;
Lundell, G. F.; Veber, D. F.; Anderson, P. S.; Chang, R.
S. L.; Lotti, V. J.; Cerino, D. J.; Chen, T. B.; Kling, P. J.;
Kunkel, K. A.; Springer, J. P.; Hirshfield, J. J. Med. Chem.
1988, 31, 2235.
Acknowledgments
We thank Norman Mason and Anne Van Abbema for
carrying out vasopressin binding experiments at Lilly,
and Elizabeth Lutz and Harlan Shannon for help with
the cyclodextrin/lactic acid formulation. This work was
supported by NIH Grants HD37290 to N.G.S. and
MH063663 to G.A.K.
17. A second potential approach is implicit in the weak
(suboptimal) binding of beta to A in Figure 1. By
screening weak binders to a related receptor (A), one
could find strong binders to the target receptor (B).
Pairwise comparisons of results for NK-2, NPY1, BK2,
and V1a screening of the Neuropeptide Cassette confirm
this hypothesis: prescreening for weak activity at any
one of the four targets resulted in 4- to 7-fold
enrichments in hit rates for the other three targets
(RFB, unpublished results). LY307174 itself had weak
but detectable affinity (IC50 ꢀ40 lM) at the hamster
NK-2 receptor.
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