Novel selective inhibitors of neutral endopeptidase for the treatment of female sexual arousal disorder

Article abstract of DOI:10.1016/j.bmc.2006.10.002

A series of substituted glutaramides were synthesised using Candoxatrilat 1 as a lead and evaluated for potency against neutral endopeptidase (NEP) as a potential treatment for female sexual arousal disorder (FSAD). In this paper, we describe studies in which we were able to increase NEP activity substantially over the levels reported for previous compounds from this programme by appropriate substitution in both the P₁^′ and P₂^′ regions. Optimisation led to the 4-chlorophenpropylamide S-30 which was selected as a candidate for further study.

Full text of DOI:10.1016/j.bmc.2006.10.002

Bioorganic & Medicinal Chemistry 15 (2007) 142–159
Novel selective inhibitors of neutral endopeptidase for the treatment
of female sexual arousal disorder
David C. Pryde,a,* Andrew S. Cook,a Denise J. Burring,a Lyn H. Jones,a Stephanie Foll,a
Michelle Y. Platts,a Vivienne Sanderson,a Martin Corless,a Alan Stobie,a
Donald S. Middleton,a Laura Foster,b Laura Barker,b Piet Van Der Graaf,b Peter Stacey,b
Christopher Kohl,c,* Sara Coggonc and Kevin Beaumontc
aDepartments of Discovery Chemistry, Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
bDiscovery Biology, Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
cPharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Ramsgate Road, Sandwich,
Kent CT13 9NJ, UK
Received 22 May 2006; revised 27 September 2006; accepted 4 October 2006
Available online 6 October 2006
Abstract—A series of substituted glutaramides were synthesised using Candoxatrilat 1 as a lead and evaluated for potency against
neutral endopeptidase (NEP) as a potential treatment for female sexual arousal disorder (FSAD). In this paper, we describe studies
in which we were able to increase NEP activity substantially over the levels reported for previous compounds from this programme
by appropriate substitution in both the P01 and P20 regions. Optimisation led to the 4-chlorophenpropylamide S-30 which was selected
as a candidate for further study.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
therapeutic agents for the treatment of FSAD through
the potentiation of vasoactive intestinal peptide (VIP)-
induced increases in genital blood flow. These acids were
based upon the Candoxatrilat (1) template,4 wherein
by targeting small (Mwt < 400), relatively polar
(0 < logD < 1) mono-carboxylic acid structures we were
able to identify several compounds which were potent
(IC50 < 50 nM), selective for NEP over related pepti-
dases and demonstrated low clearance (Cl) and volume
of distribution (Vd) in animal models. Most notably,
the amino-thiadiazole R-2 was shown to possess the best
combination of potency and a pharmacokinetic profile
suitable for prn or on-demand dosing (short elimination
T1/2, rapid onset of action), and was selected as the pro-
totype clinical candidate from the programme (Fig. 1).5
Female sexual arousal disorder (FSAD)ꢀ is believed to
be a highly prevalent sexual disorder affecting significant
numbers of pre-, peri-, and post-menopausal women.1 It
has been suggested that the most common cause of
FSAD is decreased genital blood flow, resulting in re-
duced vaginal, labial and clitoral engorgement.2 In the
previous papers in this series,3 we introduced a series
of mono-carboxylic acid glutaramide inhibitors of the
Zn-dependent metallopeptidase Neutral Endopeptidase
[NEP, EC (3.4.24.11)] which we targeted as potential
Keywords: Neutral endopeptidase; NEP; Glutaramides; FSAD.
*
Corresponding authors. Tel.: +44 1304 643687; fax: +44 1304
651819; e-mail addresses: David.Pryde@Pfizer.com; Christopher.
Kohl@Pfizer.com
In this paper, we describe further explorations of the
SAR of this series of small glutaramides using this strat-
egy, within which we sought a back-up agent to R-2
which was differentiated by a significant, ideally >10-
fold increase in NEP activity by appropriate substitu-
tion in both the P01 and P20 regions while largely retaining
the basic Candoxatrilat template. The SAR optimisation
which led to the 4-chlorophenpropylamide (S-30) is
described below.6
ꢀ
Abbreviations used: FSD, female sexual dysfunction; FSAD, female
sexual arousal disorder; VIP, vasoactive intestinal peptide; NEP,
neutral endopeptidase; ACE, angiotensin converting enzyme; ECE,
endothelin converting enzyzme; WSCDI, (1-[3-(dimethylamino)pro-
pyl]-3-ethylcarbodiimde); DEAD, 1,1-diethylazodicarboxylate;
DPPA, diphenylphosphoryl azide; LDA, lithium diisopropylamide;
HOBt, 1-hydroxybenzotriazole; n-Hept, n-heptyl; n-Pent, n-pentyl;
NMM, N-methylmorpholine.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.002

D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
143
Monocarboxylic acid
targets
OMe
Me
Y
O
H
H
N
N
H
S
X
HO2C
HO2C
N
HO2C
O
N
O
Me
N
O
1, Candoxatrilat
MWt. < 400
0< LogD >+1
CO2H
R-2
Figure 1. Lead structures and medicinal chemistry strategy.
2. Synthetic chemistry
DCM as solvent at room temperature was sufficient
to achieve complete reaction. For less active amines
(e.g., amino-substituted heterocycles) DMF or aceto-
nitrile as solvents at elevated temperatures were
required for good yields of the coupled products.
The ester protecting group could then be removed
using standard protocols to deliver the requisite acids
in excellent yields.
The glutaric acid template 3 is easily constructed by
alkylation of cyclopentane carboxylic acid dianion
(Scheme 1).3 Coupling of commercially available
and custom-synthesised amines to this acid was then
performed using WSCDI with NMM as a base.
For reactive amines (e.g., primary alkyl amines)
O
Br
X
X
H
i-ii
vi-vii
O
OH
PGO
N
R1
HO2C
O
O
O
O
3a; PG=tBu, X=H
3b; PG=tBu, X=allyl
3c; PG=tBu, X=nPr
OH
S
2; X=nPr, R1=
PG= Bn or tBu
Me
N
N
3d; PG=tBu, X=CH2OMe
S
3e; PG=tBu, X=CH2CH2OMe
3f; PG=Bn, X=CH2CH2OMe
14; X=CH2CH2OMe, R1=
15; X=nPr, R1=
Ph
N
N
Ph
iii-v
Ph
16; X=CH2OMe, R1=
Ph
O
O
17; X=CH2CH2OMe, R1=
O
O
Me
18; X=CH2CH2OMe, R1=
19; X=CH2CH2OMe, R1=
N
HO
nPent
Ph
22; X=CH2CH2OMe, R1=
25; X=CH2CH2OMe, R1=
27; X=CH2CH2OMe, R1=
OMe
nHept
28; X=CH2CH2OMe, R1=
29; X=CH2CH2OMe, R1=
Ph
Scheme 1. Reagents and conditions: (i) 3a; 2 equiv LDA, THF, ꢀ20 ꢁC ! room temperature, 16 h, 29%; (ii) 3b; 2 equiv LDA, allyl bromide, THF,
ꢀ78 ꢁC ! room temperature, 16 h, 92%; 3c; then 15 psi H2, EtOH, 10% Pd/C, room temperature, 1 h, 91% OR 3e; 2 equiv LDA, bromooethylmethyl
ether, THF, ꢀ78 ꢁC ! room temperature, 16 h, 55% OR 3d; 2 equiv LDA, chloromethyl methyl ether, THF, ꢀ78 ꢁC ! room temperature, 16 h,
66%; (iii) bromoethylmethyl ether, NaH, THF, room temperature ! reflux, 16 h, 70%; (iv) KOH, water, dioxan, rt, 16 h, then (HCHO)n, piperidine,
pyridine, 60 ꢁC, 2.5 h, 46%; (v) cyclopentane carboxylic acid, 2 equiv LDA, THF ꢀ78 ꢁC ! room temperature, 5 h, 34%; (vi) R1NH2, WSCDI,
HOBtÆNMM, solvent (see text), 25–100 ꢁC, 12 h, 38–92%; (vii) TFA/DCM (1:1), room temperature, 6–16 h or HCl (g), Et2O, DCM or EtOAc, room
temperature, 10 min, 12–97%.

144
D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
i
ii-iii
R
R
R
EtO2C
HO2C
5
4a; R=Cl
4b; R=OMe
4c; R=F
6
iv
X
HO2C
R
Scheme 1
R
ClHH2.N
N
H
O
7
20; X=CH2OMe, R=Cl
21; X=CH2CH2OMe, R=Cl
23; X=CH2CH2OMe, R=OMe
24; X=CH2CH2OMe, R=F
Scheme 2. Reagents and conditions: (i) EtO2CCHN2, Rh2(OAc)4, toluene, 1 h, 85 ꢁC, 31–41%; (ii) NaOMe, EtOH, 85 ꢁC, 18 h; (iii) LiOH (aq),
t
MeOH, 70 ꢁC, 16 h, 78–96% over 2 steps; (iv) DPPA, NEt3, BuOH, 90 ꢁC, 48 h, 80–89%.
Hal=Br or I
X
HO2C
i-ii
Scheme 1
R
R
H2N
Hal
N
H
R
O
9
8a; R=OMe
8b; R=F
30; X=CH2CH2OMe, R=Cl
31; X=CH2CH2OMe, R=F
32; X=CH2CH2OMe, R=OMe
Scheme 3. Reagents and conditions: (i) acrylonitrile, P(o-Tol)3, Pd (OAc)2, NEt3, MeCN, 80 ꢁC, 14 h, 45–56%; (ii) NH3 (aq), EtOH, 40 psi H2, Ra–
Ni, 25 ꢁC, 12 h, 43–88%.
OMe
OMe
60psi H2
BnO2C
HO2C
10wt% Pd/C
EtOH
N
N
O
H
16h
90%
O
H
10
26
Scheme 4.
O
OMe
H2N
N
HO2C
O
i-ii
Scheme 1
OH
O
11
O
13
N
H
O
OMe
OMe
OMe
33
HO
12
Scheme 5. Reagents and conditions: (i) DEAD, PPh3, DCM, 25 ꢁC, 16 h, 50%; (ii) hydrazine hydrate, methanol, reflux, 4 h then 25 ꢁC, 48 h, 63%.
Chiral, non-racemic versions of the acids 3 were ob-
tained from the racemic material using a (+)-pseudo-
ephedrine-mediated classical resolution procedure7
described in Section 5. Non-commercial and novel
amines were prepared by the procedures described in
Schemes 2–5. Thus, 1-amino-2-aryl cyclopropanes 7

D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
145
Table 1. Dog NEP activity for various substituted glutaramides
were prepared by an initial cyclopropanation catalysed
by rhodium acetate dimer, and then equilibrated and
hydrolysed to the most thermodynamically stable
trans-1-carboxyl-2-aryl cyclopropanes 6. A Curtius rear-
rangement provided the aminocyclopropanes 7, which
were then coupled to the acids 3 according to the proce-
dure of Scheme 1.
X
H
N
R1
HO2C
O
Compound
X
R1
dNEP IC50
(nM)
Several phenpropylamines were prepared by Heck reac-
tion (Scheme 3) of the corresponding aryl halides 8 with
acrylonitrile, followed by reduction with Raney nickel to
the requisite amines 9 and coupling to the acids 3. In the
case of p-chloroiodobenzene, the reduction step was
accompanied by some dechlorination, and so an alterna-
tive literature method of preparing this example of
amine 9 was used.8
S
S
2
14
15
16
17
18
nPropyl
60
Me
Ph
N
N
Methoxyethyl
nPropyl
5
160
100
65
N
N
Ph
The a-methyl phenethylamine 26 was made by
simple hydrogenation of the benzyl ester analogue
10.
Methoxyethyl
Methoxymethyl
Methoxyethyl
Ph
Ph
The phenoxyethylamine 33 was prepared by the 4 step
sequence outlined in Scheme 5. N-hydroxyethyl phthal-
imide 11 underwent a smooth Mitsunobu reaction with
4-methoxy phenol 12. Deprotection and coupling
according to Scheme 1 provided the phenoxy compound
33 in good yield after deprotection.
Bu
17
N
19
Methoxyethyl
11
HO
3. Results and discussion
aMean data of at least two determinations.
bAll entries are racemic mixtures.
cAll compounds showed >3 lM activity versus pACE.
Compounds were first evaluated in vitro as inhibitors of
canine NEP (dNEP) using a spectrophotometric assay,
involving the cleavage of Abz-D-Arg-Arg-Leu-EDDnp
to generate the fluorescent product Abz-D-Arg-Arg.9
Soluble NEP was obtained from the kidney cortex by
an adaptation of a literature method.10 Selectivity over
the related peptidases porcine angiotensin converting
enzyme (pACE) and human endothelin converting en-
zyme (hECE-1) was established using Abz-Gly-p-nitro-
Phe-Pro-OH and Big ET-1 as substrates, respectively.
Potent and selective compounds were assayed against
other NEP species, including human NEP (hNEP), ob-
tained from human kidney, and progressed into in vitro
metabolic stability assessment (including logD and
Caco-2 cell permeability measurement) and in vivo
pharmacokinetic evaluation; promising compounds
which emerged from this analysis were assessed for effi-
cacy in an animal model of genital blood flow11 the re-
sults of which will be described elsewhere.
effects of these modifications were further investigated
through retaining a phenyl-containing P02 group, but
this time using a cyclopropane ring as an alternative
linker to the thiadiazole. The trans-2-phenyl cyclopro-
pyl-containing template 15 is 160 nM against dNEP
with an n-propyl P01. The same feature in a methoxy-
ethyl-containing molecule 16 is slightly more potent at
100 nM. A methoxymethyl group 17 improved poten-
cy slightly, but raised the possibility of b-elimination
of methanol through removal of the proton adjacent
to the acid. Nonetheless, it was clear that larger bulk
adjacent to the amino-cyclic group, provided an
appropriate linking scaffold was applied, or oxygena-
tion of the a-carboxyl side chain could offer potency
gains. A similar trend was seen in both the amino-
pyridine series of 18, where a methoxyethyl side chain
gave a 30-fold improvement in potency over propyl,
and in the indanol series 19, where the same change
provided a 3-fold potency gain. We viewed the cyclo-
propane structures in particular as being quite differ-
ent to the amino heterocycles such as 2 and as
offering quite a bit of scope for further elaboration
and potency optimisation. These were, therefore,
followed up as top priority.
3.1. Amide SAR
The racemic version of the ethyl aminothiadiazole 2 is
a potent and selective inhibitor of dog NEP with an
IC50 of 60 nM (Table 1).3 Increasing the size of the
ethyl substituent to a benzyl group was proposed to
increase potency through making additional hydro-
phobic contacts, albeit with an incumbent increase
in lipophilicity. Therefore, an oxygen atom was also
inserted into the P01 propyl side chain to modulate
the logD of this target. Compound 14 did indeed
prove to be some 10-fold more potent than 2. The
3.2. Cyclopropanes
The trans-2-phenyl cyclopropane moiety of 15–17
showed reasonable potency in the 100 nM region,

146
D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
similar to the starting point 2. The methoxyethyl P01 sub-
stituent was seen as one of the most versatile side chains,
offering good potency in a relatively low lipophilicity
structure, and was used to explore a number of cyclo-
propane ring substituents, including halogens, methoxy
and an extended pentyl chain (Table 2). The chlorophe-
nyl group was also made in the methoxymethyl P01 series
for comparison. In fact, the activity of the two P01 series
in this case was identical. Threefold to fourfold potency
gains were made by substituting a halogen atom on the
4-position of the benzene ring. Most successful was
substituting with a methoxy group which gave an
approximately 10-fold gain in potency, as did the
extended n-pentyl substituent. The clear message in this
series was that potency gains could be made by extend-
ing functionality outwards from a suitably orientated
amino-cyclic group. The origin of these potency gains
is a better fit of these functionalities into a channel with-
in the S02 binding region, as evidenced by co-crystal
structures of a variety of project compounds in a human
NEP construct. A full account of this aspect of the pro-
ject will be disclosed, elsewhere. Preparation of the ami-
no-cyclopropanes required a rather lengthy synthesis,
and ready access to chiral, non-racemic versions of both
cis- and trans-amino-cyclopropanes was not trivial. We
were also concerned about the chemical stability of these
1-amino-2-aryl-cyclopropanes and their potential to
form reactive intermediates.
groups, which were structurally very different to the
majority of the potent compounds we had encountered
up to that point, but would still be able to access confor-
mations and, therefore, activities of the cyclopropane
analogues from which they were derived.
3.3. Aliphatic amides
Deleting the central cyclopropane ring carbon atom in
16 produced a 5-fold drop in binding potency, 25
(Table 3). Reducing the cyclopropane ring to produce
an a-methyl phenethylamine 26 gave a similar drop in
potency. Even reinstalling the most potent phenyl ring
substituent, namely methoxy, into the phenethylamine
template 27 did not alter potency greatly. Breaking open
the cyclopropane ring of the pentyl-cyclopropane 28
largely retained potency, and hinted at there being much
more volume within P01 binding site to be accessed,
which a phenethyl group was simply not reaching into.
This assertion was borne out by extending the pheneth-
ylamine to a phenpropylamine 29 which improved
potency by approximately 15-fold. Repeating the phenyl
substitutions which had been successful in the case of
the cyclopropanes above again gave excellent improve-
Table 3. Dog NEP activity of aliphatic glutaramides
OMe
H
N
Small variations to the aminocyclopropanes which
would produce much more synthetically accessible and
chemically more robust targets were therefore investi-
gated. The simplest and most attractive option was to
break open the ring. This would produce aliphatic amide
R1
HO2C
O
Compound
R1
dNEP IC50 (nM)
500
25
Ph
Table 2. Dog NEP activity of cyclopropyl glutaramides
Me
26
27
28
29
30
31
32
33
426
369
8
X
H
Ph
N
R1
HO2C
OMe
O
Compound
X
R1
dNEP
IC50 (nM)
20
21
22
23
24
Methoxymethyl
Methoxyethyl
Methoxyethyl
Methoxyethyl
Methoxyethyl
20
21
10
8
Cl
Cl
35
2
Cl
F
15
2
OMe
F
OMe
OMe
40
35
aMean data of at least two determinations.
O
bAll entries are racemic mixtures at both the P10 centre and at the
cyclopropane ring junctions. All compounds are trans
diastereoisomers.
aMean data of at least two determinations.
bAll entries are racemic mixtures at the P01 centre.
cAll compounds showed >3 lM activity versus pACE.
cAll compounds showed >3 lM activity vs. pACE.

D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
147
ments in potency. p-Chloro 30 and p-methoxy 32 were
again particularly potent, although we did not favour
the methoxy compound for fear of metabolic instability.
The p-fluoro analogue 31 was some 7-fold less active
than these compounds. Inserting an oxygen atom into
the benzylic position of the propyl linker in 33 gave a
drop of some 17-fold potency. These results indicated
that the phenpropyl group occupied the binding site of
NEP very much more effectively than shorter or smaller
amide substituents, or heteroatom-linked substituents
and that by appropriate substitution of the phenyl ring,
large gains in binding potency could be realised. Very
quickly we had arrived at low-nanometer potency, with-
in the range of our criteria for a back-up agent to 2.
P01 groups, and S for alkoxyalkyl groups, for example,
methoxyethyl, both of which have the same stereochem-
ical sense). A range of compounds from the above tables
were therefore synthesised as single enantiomers and
their activity against canine NEP confirmed (Table 4).
Coupling of the corresponding phenpropyl amines to re-
solved versions of the acids 3 provided the single enan-
tiomers of 2, 19, 30, 23 and 31. These compounds
were indeed found to be significantly more active than
the racemic mixtures, and again the phenpropyl com-
pounds remained the most potent examples. Interesting-
ly, in the case of the fluoro analogue S-31, the ratio of
racemate potency to single enantiomer potency (7.5)
was significantly greater than for the other two com-
pounds. All compounds were shown to be completely
selective for NEP over the related metallopeptidases
ACE and ECE-1.
3.4. Chiral, non-racemic compounds
Previous inhibitors from this series showed stereoselec-
tive binding, in favour of the R-enantiomer3 (R for alkyl
3.5. In vitro ADME data
Table 4. Single enantiomer potency for selected compounds
All compounds from the above table were also pro-
gressed into a battery of in vitro tests to assess in vitro
stability, permeability and physical properties, and com-
pared with those of R-2 (Table 5 below).
X
H
N
R1
HO2C
O
These data are interesting for a number of reasons. They
offered a good range of compound properties with
which to check our initial assertions in the project that
a logD between 0 and 1, and Mwt below 400 would pro-
vide the best chance of achieving a good absorption pro-
file. There is a relatively linear relationship between
logD and Caco-2 permeability; setting a Caco-2 perme-
ability of a compound which is predicted to be well ab-
sorbed in vivo at approximately 5, the data in the table
suggest a minimum logD of ca. 0. This obviously distin-
guishes the polar S-19 as poorly permeable and would
therefore not be expected to be well absorbed in vivo.
All compounds are very stable in HLM, although our
assay conditions did not allow us to quantify the glucu-
ronidation we expected in this series of carboxylic
acids.12 Table 5 clearly identifies the two halophenyl
compounds S-30 and S-31 as the highest fluxed, and
the most promising agents for progression. S-23 is also
well-fluxed, and interestingly our initial fears of the po-
tential lability of the OMe group were not borne out by
Compound
X
R1
dNEP IC50 (nM)
23.7
S
R-2
Me
Me
N N
S-19
S-30
OMe
OMe
5.4
0.9
HO
Cl
S-23
S-31
OMe
OMe
8
2
OMe
F
aMean data of at least two determinations.
bAll compounds showed >3 lM activity versus pACE and hECE-1.
Table 5. In vitro data for selected compounds
Compound
Mwt
logDa
Human PPB (%)b
HLM (min)c
Caco-2 Papp
(·10ꢀ6 cmsꢀ1 d
)
A–B
B–A
R-2
339
389
396
392
379
0.5
ꢀ0.2
1.0
97.9
77.2
98.3
96.8
87.6
>120
>120
>120
>120
>120
9
1
14
S-19
S-30
S-23
S-31
2.5
14
12
7
0.3
0.5
ND
ND
12
a All logD measurements were made at pH 7.4 in n-octanol and phosphate buffer.
b Human plasma protein binding was determined in vitro at 1 lg/ml by equilibrium dialysis.
c Metabolic stability was determined in human liver microsomes at 1 lM compound concentration and 0.5 lM CYP concentration. The data are
expressed as the first-order compound disappearance half-life.
d Caco-2 apparent permeability (Papp) was determined at pH 7.4 in both apical and basolateral chambers.

148
D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
the HLM data for this compound. S-19 was considered
the least attractive compound because of its poor flux,
but as will be seen below, low permeability in the
Caco-2 model at pH 7.4 did not rule out complete in vivo
absorption in this series of compounds.
additional variation in binding potency across these
species (Table 7).
In the case of R-2, there is an approximately 2.6-fold
maximum variation between the different species assay
results which could be considered as being within the as-
say variability. For the second, aliphatic amide contain-
ing compound S-30, the variation is much bigger at
approximately 8.6-fold at its maximum. Again, it seems
that aliphatic amide groups are most prone to species
variability.
3.6. pH differences and human NEP translation
The primary amino acid sequence of human NEP is
>90% identical to that of other mammalian species,
where the sequence is known. Sequence conservation is
highest around the region which makes up the active site
and at the outset of this work, we did not predict any
particular differences in activity of our series of glutara-
mides in dog vs. human-derived NEP. Indeed, the first
several compounds which were tested against both spe-
cies of NEP showed very similar IC50 values such as
R-2 and S-19. It is notable that all compounds which
feature an aliphatic or small cyclic amide substituent
show similar or weaker potency against hNEP than
against dNEP (S-30, 23 and 31), while compounds
which show greater potency against hNEP feature a
larger cyclic group attached to the amide N atom.
During the course of these investigations into species
differences, we had cause to look very carefully at the
precise assay conditions which were being used to assess
the potency of our compounds. The initial assay condi-
tions used a pH of 7.0. Assay conditions were modified
to produce a solution of pH 7.4 and the compounds of
Table 6 were tested again for NEP activity in both hu-
man and dog assays (Table 8).
We found immediately that all compounds, without
exception were less active in the pH 7.4 assay system
than at pH 7.0. Typically, compounds were approxi-
mately 2–5 times less active at pH 7.4 than at pH 7.0.
Upon closer inspection however, we noted that the drop
in potency was mirrored across species, and had little
effect on the rank order of potency, that is, the most
potent compounds at pH 7.0 were still among the most
potent at pH 7.4. In other words, the primary screening
protocol which we used to prioritise compounds to be
examined in our functional model of sexual arousal
was unaffected by the pH variations. Figure 2 shows a
plot of the canine and human NEP IC50’s at the 2
pH’s for a series of monoacid NEP inhibitors (not all
data points are represented by the structures in Table
8; some are not shown in this paper).
Clearly, those compounds which feature a larger cyclic
group appended to the amide N atom will occupy differ-
ent space and make a different set of interactions to the
aliphatic compounds in the immediate vicinity of the
amide linkage, which is possibly manifest as variant
activity against the two NEP species.
R-2 and S-30 were further assayed against rat, rabbit
and recombinant hNEP to determine the extent of any
Table 6. Comparison of hNEP and dNEP IC50’s for selected
compounds
Compound
hNEP IC50 (nM)
dNEP IC50 (nM)
R-2
18.9
3.7
2.3
6.1
4.6
23.7
5.4
0.9
1.5
4.3
There is an overall parallel shift of the data points away
from the central line of complete correlation, indicating
a consistent shift in IC50 values. In the case of hNEP, the
shift was of the order of some 4.6-fold, whereas for dNEP
it was about 2.8-fold. This effect has been noted before for
NEP,13 and for other metalloproteases such as ECE.14
S-19
S-30
S-23
S-31
aMean data of at least two determinations.
Table 7. IC50 of selected compounds against several species of NEP
Compound
HNEP IC50 (nM)
DNEP IC50 (nM)
ratNEP IC50 (nM)
rabbitNEP IC50 (nM)
rec-hNEP IC50 (nM)
R-2
S-30
18.9
2.3
23.7
0.9
14.3
0.6
10.2
0.8
19.7
5.4
aMean data of at least two determinations.
Table 8. Comparison of hNEP and dNEP IC50’s at pH 7.0 and 7.4
Compound
pH 7.0
pH 7.4
hNEP IC50 (nM)
dNEP IC50 (nM)
hNEP IC50 (nM)
dNEP IC50 (nM)
R-2
18.9
3.7
2.3
6.1
4.6
23.7
5.4
0.9
1.5
4.3
31.8
10
ND
14.2
3.8
S-19
S-30
S-23
S-31
10.5
13.8
12.6
5.7
8.8
aMean data of at least two determinations. ND, not determined.

D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
149
Explanations of these pH differences are usually cited as
80
60
40
20
0
being the difference in the extent of ionisation of the
inhibitor or the enzyme. In the case of this series of
inhibitors, the acid functional group has pKa values of
approximately 4.6 with little differences across the series,
offering significantly more than 99% of ionised com-
pound at both pH 7.0 and 7.4. The minute difference
in ionised compound at pH 7.0 and 7.4 would not be
expected to account for the differences seen above, un-
less uncharged compound contributed to NEP inhibi-
tion. The increase by 0.4 pH units reduces the amount
of unionised compound by more than 2-fold. On the
other hand, the active site of NEP does contain several
highly basic residues, and also two histidine residues
and a glutamic acid in the crucial vicinity of the zinc
atom, and it seems to be more likely that small changes
in the enzyme ionisation would better account for the
observed potency differences, although evidence for this
is lacking.15 Since IC50 values for competitive inhibitors
are dependent on the substrate concentration used in the
assay relative to substrate affinity (Km), the large in-
crease in IC50 with increasing pH could potentially be
explained if the affinity for the substrate significantly
increased with increasing pH. Our observations however
(data not shown) do not support this explanation, since
at pH 7.0, 7.4 and 8.0 we found the Km to be >100 lM,
which is at least 5 times the substrate concentration we
chose for the IC50 experiments. Interestingly, our Km
estimate for the NEP substrate is significantly higher
than the original published value of 2.8 lM.9 Explana-
tions for this might include a batch variation between
the published peptide and the version we synthesised,
or potential inaccuracies in determining the Km for fluo-
rescent substrate of this type, which due to fluorescence
inner filter effects can be distorted to appear lower than
the true value. The effect of pH on the IC50 of S-30
across a much bigger pH range was also examined.
Across the range of Tris buffer (approximately pH
6.5–8.5), the IC50 was seen to increase with increasing
pH in an apparently exponential fashion (Fig. 3).
6.8
7.0
7.4
8.0
8.6
pH
Figure 3. Potency variation of S-30 with increasing pH (values are as
follows: pH 6.8, IC50 1.8 nM; 7.0, 2.9; 7.4, 5.2; 8.0, 15.0; 8.6, 70.9).
and/or exercise would not be expected to fall below this
value by very much.
The assays described above consistently provide a rank
order of compound potency at any given pH, to allow
the selection of compounds for progression into animal
models of sexual arousal and pharmacokinetic evalua-
tion. Therefore, while worth noting, this phenomenon
made no practical differences to the project.
3.7. Metabolic profile
Several compounds were evaluated in the rat, and their
pharmacokinetic profiles measured (Table 9). All com-
pounds displayed short T1/2 and were also low Cl with
high bioavailability. All compounds showed rapid
absorption, with a Tmax in each case of <1 h, that is,
an ideal pharmacokinetic profile for prn dosing. This
included S-19, which despite its polarity, and poor
Caco-2 flux was well absorbed, which called into ques-
tion the value of the Caco-2 model in predicting in vivo
absorption in this series of compounds.16
It will be seen however, that across the compound range,
there is some considerable variation in Vd, which is not
expected from the physicochemical properties. We offer
two reasonable hypotheses to explain this phenomenon.
First, reversible metabolism (Fig. 4) may contribute to
the unusual pharmacokinetic profiles observed.17
The key issue, of course, was exactly what the most
physiologically relevant pH was at which to measure
compound potency. Our take on these observations
was that the most relevant pH for this indication will
most probably be pH 7.4. The pH of normal, healthy
vasculature is 7.4, and even under conditions of stress
Reversible metabolism occurs when a metabolite or bio-
transformation product and the parent drug undergo
interconversion in both directions. All representatives
of the glutarate series profiled underwent acyl glucuron-
idation. The acyl glucuronide can be excreted via the bile
HumanNEP
Canine NEP
-5
-5
-6
-7
-6
-7
-8
-8
-9
-9
-10
-10
-10
-9
-8
-7
-6
-5
-10
-9
-8
-7
-6
-5
Log10 IC50 pH7.0
Log10 IC50 pH 7.0
Figure 2. Comparison of pH 7.0 and 7.4 IC50’s in human and dog NEP.

150
D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
Table 9. Pharmacokinetics in rat of selected compounds
Compound
Mwt
logD pH 7.4
Cl (ml/min/kg)
Vd (L/kg)
T1/2 (h)
fu
F (oral) (%)
R-2
339
389
396
379
0.5
ꢀ0.2
1.0
3.0
18
0.44
7.5
0.6
2.7
1.7
4.8
1.9
2.4
0.106
0.088
0.083
0.138
103
109
70
S-19
S-30
S-31
3.5
13
0.5
138
aJugular vein cannulated rats were dosed at 1 mg/kg iv (tail vein) and po (gavage). Blood samples were withdrawn via the indwelling catheter until
24 h post-dose.
ronidation capacity (such as dog19) and in species with a
low threshold for biliary excretion (such as rat and
dog20) the apparent clearance will particularly underes-
Acyl
Glucuronide
R-COOH
timate the true elimination capacity. The pharmacoki-
netics of those carboxylic acids that are predominantly
glucuronidated (such as S-30 and S-31) will suffer signif-
icantly more from this complication than compounds
with minute to moderate acyl glucuronidation. In hu-
mans, where the biliary threshold tends to be higher
and the glucuronidation capacity is usually lower than
in dog, reversible kinetics will have less of an influence.
Similar considerations can be made for the second pri-
mary PK parameter, the volume of distribution (Vd).
Figure 4. Reversible metabolism of carboxylic acids.
into the gut, where it will be chemically and/or enzymat-
ically cleaved. The liberated parent is available for reab-
sorption (entero-hepatic cycling). It is likely that the
secretion of the acyl glucuronide into the bile is medi-
ated by MRP2, a carrier protein with high affinity to
drug glucuronides.18 Some acyl glucuronide will escape
the biliary pathway and appear in the blood. There, it
may be rapidly degraded and release parent depending
on its chemical stability and its affinity to plasma ester-
ases. For example, the decomposition half-life of the
acyl glucuronide of R-2 is ca. 5 min in dog, rat and hu-
man blood, whereas the acyl glucuronide of S-30 does
not degrade in blood from these species during 1 h at
37 ꢁC. A fuller account of the profile and behaviour of
the acyl glucuronides of this series of compounds will
be reported elsewhere.
Second, active concentrative hepatic uptake of com-
pounds can lead to a volume of distribution larger than
expected from their physicochemistry.21 This is particu-
larly evident for acidic compounds. For example, the
acidic NSAID ketoprofen is actively taken up into rat
liver22 which results in a volume of distribution between
0.8 and 1.1 L/kg,23 considerably larger than the 0.2 L/kg
expected from physicochemistry. Similar observations
have also been made for a dicarboxylic acid endothelin
receptor antagonist.24 Taking into account these acid-
specific phenomena, S-30 was chosen to further progress
into a model of sexual arousal on the basis of low un-
bound clearance and potency. These results will be pre-
sented in due course.
Hence, the pharmacokinetics of the glutaramides can be
confounded by these two cyclic (reversible) processes.
The irreversible clearance (Cl10; cf. Fig. 5) of a glutarate
can be achieved by non-UGT (i.e., CYP) mediated
metabolism or by renal clearance of parent and/or acyl
glucuronide (Cl20).
4. Conclusion
We have shown that relatively small (Mwt < 400) substi-
tuted phenpropylamides with halide or alkoxy substitu-
ents are potent and selective inhibitors of NEP.
Incorporation of various substitution patterns at the
P01 position is tolerated, and changes in this region, along
with changes in the substituents on the phenpropyl-
amine fragment, allowed a range of compounds to be
prepared with disparate logD. Profiling of some of these
compounds in animal models has suggested these NEP
inhibitors should be efficacious with a pharmacokinetic
profile which is suited to prn dosing in humans. Of these
compounds, S-30 was chosen for further study.
From the above it is clear that the basic pharmacokinet-
ic relationship Cl = Dose/AUC will provide only an
apparent clearance value. This value will always under-
estimate the total elimination capacity for the drug, that
is, Cl10 + Cl12. The apparent clearance value observed
will be the more inaccurate, the greater the extent of
Cl12, that is, the greater the contribution of acyl glucu-
ronidation to total clearance. In species with high glucu-
Cl12
Cl21
Cl20
Cl10
Parent in
body
Acyl Glucuronide
in body
K41
Cl23
5. Experimental
5.1. Chemistry
K34
Parent in
intestine
Acyl Glucuron-
ide in intestine
Melting points were determined on a Gallenkamp Melt-
ing point apparatus using glass capillary tubes and are
uncorrected. Unless otherwise indicated all reactions
Figure 5. Clearance terms and rate constants in the PK of carboxylic
acids.

D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
151
were carried out under a nitrogen atmosphere, using
commercially available anhydrous solvents. Thin-layer
chromatography was performed on glass-backed pre-
coated Merck silica gel (60 F254) plates, and flash chro-
matography was carried out using 40–63 lm silica gel.
Proton NMR spectra were measured on a Varian Inova
300 or 400 spectrometer in the solvents specified. Mass
spectra were recorded on a Fisons Trio 1000 using ther-
mospray positive (TSP) ionisation. Combustion analy-
ses were conducted by Exeter Analytical UK. Ltd,
Uxbridge, Middlesex. The purity of compounds was
also carefully assessed using analytical TLC and proton
NMR, the latter technique was used to calculate the
amount of solvent in solvated samples. In multistep
sequences, the purity and structure of intermediates
were verified spectroscopically by 1H NMR. Optical
rotations were determined using a Perkin-Elmer 341
polarimeter. Optical purity was determined by analytical
HPLC analysis of classically resolved material (see Sec-
tion 5 below) via comparison to racemic material. Un-
less otherwise stated, all starting materials and solvents
were readily available from commercial suppliers. The
preparation of compounds 2, 14, 18 and 19 has been de-
scribed previously.3b Compounds 15–17, 22, 25 and 27–
29 were all prepared from commercially available
amines. Compound 9 (R = Cl) was prepared by the liter-
ature method.9
The NEP assay was highly reproducible. We demon-
strated this from a statistical analysis of 25 IC50 values
obtained from a single compound tested multiple times
over an 8-month period. By assessing the assay-to-assay
variation we were able to conclude that two replicates
would be sufficient to ensure that the geometric mean
IC50 was estimated with less than 2-fold error, based
on the width of the 95% confidence interval.
5.3. ACE inhibition assay
Performed as described above for the NEP assay, except
the substrate used was o-aminobenzoyl-Gly-Gly-p-nitro-
Phe-Pro-OH, at a final assay concentration of 10 lM,
the enzyme and substrate buffer was 50 mM Tris–Cl,
pH 7.4 (at 37 ꢁC), 300 mM NaCl, and the reaction was
terminated using 100 ll of a 2 mM EDTA solution.
5.4. ECE-1 inhibition assay
Performed as described above for the ACE assay.
5.5. Pharmacokinetic studies
Male CD rats (Charles River, Manston, UK) were used.
These were jugular vein cannulated under general anaes-
thesia, with the cannula exteriorised at the back of the
neck. At specified times after dosing, 0.2 ml samples of
blood were withdrawn from the cannula and transferred
to heparin tubes. Following adequate mixing the blood
samples were centrifuged and plasma was generated.
Immediately after that plasma was stabilised by addition
of 0.01 parts of phosphoric acid (85%, v/v) and stored
deep frozen until analysis. The fraction unbound in rat
plasma was determined at 1 lg/ml using equilibrium
dialysis against isotonic Krebs–Ringer bicarbonate buff-
er (pH 7.4). The concentrations of glutaramides in rat
plasma were determined by LC–MS–MS (Hewlett Pack-
ard HP1100 binary HPLC pump, CTC Pal Autosam-
pler, Sciex API 2000 mass spectrometer with
TurboIonSpray interface) at a flow rate of 1 ml/min,
split 50:1 post-column using an AcurateTM flow splitter.
Glutaramides and internal standard (see Table 10) were
extracted from 100 ll of acidified plasma mixed with
400 ll of 50 mM ammonium formate buffer (pH 3.5)
using activated C18 IST cartridges in 96-well format
(Porvair). The cartridges were washed with 1 ml of
50 mM ammonium formate (pH 3.5). Compounds were
eluted with 1 ml of acetonitrile containing 5% (v/v) for-
mic acid and evaporated to dryness under nitrogen at
20 ꢁC. The residues were resuspended in 200 ll of
2 mM ammonium acetate in 70:30 (v/v) methanol/water
(pH 3.5) and 180 ll was injected into the HPLC-mass
spectrometer. The mobile phase was 2 mM ammonium
acetate in methanol/water (90:10; v/v; pH 3.5) and
5.2. NEP inhibition assay
In a 96-well microtitre plate 100 ll of fluorescent NEP
substrate peptide (50 lM o-aminobenzoyl-d-Arg-Arg-
Leu-ethylenediamine 2,4-dinitrophenyl) was added to
50 ll of 4 times test concentration (4·) inhibitor solu-
tions. The assays were initiated by the addition of
50 ll of enzyme which had been previously diluted such
that with an incubation time of 1 h the percentage of
substrate converted to product was approximately 10%
or less. The reaction mixture was incubated for 1 h at
37 ꢁC in a shaking incubator, then stopped by the addi-
tion of 100 ll of 300 nM phosphoramidon. Samples cor-
responding to 100% substrate to product conversion
were included to enable the percent substrate proteoly-
sed to be determined. Each assay point was in duplicate.
Each IC50 experiment used a 10 point dilution series at
half-logarithmic inhibitor concentration increments,
with the highest concentration either 10, 1 or 0.1 lM.
After the 1 h incubation, fluorescence of the samples
was measured (Ex320/Em420) using a BMG Fluostar
Galaxy reader and an IC50 calculated using a customised
Microsoft Excel add-inn. Enzyme and substrate were
prepared in 50 mM Tris–Cl (pH 7.4 at 37 ꢁC), and 4·
inhibitor dilutions were made in 50 mM Tris–Cl, pH
7.4, at 37 ꢁC/4% DMSO. In the experiments where the
pH of the assay was modified, enzyme, substrate and
inhibitor solutions were all prepared in Tris buffer at
the appropriate pH. The range of pH validated pre-set
buffer crystals (Sigma, UK) was used for this. Standard
compounds assessed in this assay were as follows; phos-
phoramidon 0.71 nM, thiorphan 3.85 nM and candoxa-
trilat 2.61 nM. IC50 estimates stated for NEP represent
the geometric mean of at least two values obtained using
separately weighed and solubilised compound samples.
the column was
a Hypersil HS100 C18, 5 lm,
50 · 4.6 mm. Detection was carried out by positive ion
multiple reaction monitoring (MRM) at Q1 and Q3 res-
olution of ca. 0.7 Da peak width at half-height (Table
10). The curtain gas, nebuliser gas and TurboIonSpray
gas was nitrogen at settings of 30 (CUR), 25 (GS1)
and 40 (GS2), respectively. TurboIonSpray temperature
was 100 ꢁC. The collision gas was nitrogen at a setting of

152
D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
Table 10. MRM transitions for the plasma analysis of glutaramides
5.6.1.4. 4-Methoxy-2-[1-(trans-2-phenyl-cyclopropyl-
carbamoyl)-cyclopentylmethyl]-butyric acid tert-butyl
ester, tert-butyl-16. dH (CDCl3, 400 MHz) 1.10–1.14
(m, 1H), 1.16–1.20 (m, 1H), 1.42 (s, 9H), 1.58–1.67 (m,
5H), 1.70–1.72 (m, 1H), 1.89–1.91 (m, 2H), 2.02–2.10
(m, 2H), 2.43 (q, J = 6.8 Hz, 1H), 2.83 (q, J = 6.8 Hz,
1H), 3.29 (s, 3H), 3.11–3.18 (m, 2H), 6.15 (br s, 1H),
7.09–7.23 (m, 5H). LRMS: (TS+) 416 (M+H). Anal.
Found C, 71.61; H, 9.01; N, 3.36%. C25H37NO4 requires
C, 71.94; H, 8.98; N, 3.36%. Yield 60%.
Compound
Q1
Q3
R-2
S-9
S-20
S-21
22
340
390
396
380
392
418
326
130
164
346
330
342
192
116
4
15
5.6.1.5. 2-Methoxymethyl-3-[1-(trans-2-phenyl-cyclo-
propylcarbamoyl)-cyclopentyl]-propionic acid tert-butyl
ester, tert-butyl-17. dH (CDCl3, 400 MHz) 1.00–1.10 (m,
2H), 1.40 (s, 9H), 1.50–1.80 (m, 6H), 1.80–2.05 (m, 4H),
2.30–2.40 (m, 1H), 2.70–2.80 (m, 1H), 3.20 (s, 3H),
3.25–3.35 (m, 1H), 3.70 (s, 3H), 6.05 (s, 1H), 6.70–7.05
(m, 5H). LRMS: m/z (TS+) 446 (M+H). Yield 62%.
2. The collision energy was 25 eV (OR 30 V), the dwell
time was 200 ms with 50 ms pause.
All other data were obtained using standard methods.
5.6. General procedure 1. Amide formation
The requisite amine derivative (1 equiv) and the acid 3a–
f3,7 (1 equiv) were dissolved in DCM or MeCN (1 M)
and NMM (1 equiv) added in one portion. HOBt (1
equiv) was then added, followed by 1 equiv of WSCDI.
The mixture was stirred at room temperature (except in
the case of less reactive amino heterocycles viz. tert-bu-
tyl-2, -14 and -18 or hindered viz. tert-butyl-19 amines
which were heated at 100 ꢁC in DMF) for 12 h under
a nitrogen atmosphere, or until reaction was complete
as judged by TLC analysis. The mixture was diluted
with EtOAc and washed several times with water. The
organic layer was separated, washed again with water,
dried (MgSO4) and evaporated to afford the desired
amides, which were purified by flash chromatography
using mixtures of either pentane and EtOAc or MeOH
and DCM (typically between 30% and 50% EtOAc in
pentane or between 2% and 5% MeOH in DCM).
5.6.1.6. 2-Methoxymethyl-3-[1-(trans-2-phenyl-cyclo-
propylcarbamoyl)-cyclopentyl]-propionic acid benzyl
ester, 10. dH (CDCl3, 400 MHz) 1.00–1.06 (m, 1H),
1.22 (dd, J = 10.4, 6.8 Hz, 1H), 1.37–1.48 (m, 2H),
1.60–2.10 (m, 10H), 2.52–2.59 (m, 1H), 2.77–2.84 (m,
1H), 3.22 (s, 3H), 3.37 (t, J = 6.5 Hz, 2H), 5.03 (s,
2H), 5.90 (s, 1H), 7.11–7.32 (m, 10H). Anal. Found C,
74.17; H, 7.90; N, 3.17%. C28H35NO4 requires C,
74.21; H, 7.87; N, 3.09%. Yield 74%.
5.6.1.7.
2-[1-(4-Butyl-pyridin-2-ylcarbamoyl)-cyclo-
pentylmethyl]-4-methoxybutyric acid benzyl ester, Bn-
18. dH (CDCl3, 400 MHz) 0.89 (t, J = 6.9 Hz, 3H), 1.24–
1.32 (m, 2H), 1.47–2.26 (m, 14H), 2.55–2.68 (m, 3H),
3.17 (s, 3H), 3.24 (t, J = 7.3 Hz, 2H), 4.99 (s, 2H), 6.83
(d, J = 5.3 Hz, 1H), 7.27–7.35 (m, 5H), 7.94 (br s, 1H),
8.07 (s, 1H), 8.13 (d, J = 5.4 Hz, 1H). Yield 66%.
The following amides were prepared as protected forms
of the final target structures according to
5.6.1.8. 2-[1-(2-Hydroxymethyl-indan-2-ylcarbamoyl)-
cyclopentylmethyl]-4-methoxy-butyric acid tert-butyl
ester, tert-butyl-19. dH (CDCl3, 400 MHz) 1.40 (s, 9H),
1.44–2.00 (m, 12H), 2.37–2.43 (m, 1H), 2.99 (d,
J = 15.6 Hz, 1H), 3.08 (d, J = 15.6 Hz, 1H), 3.20–3.38
(m, 7H), 3.65 (dd, J = 6.8, 0.8 Hz, 2H), 4.40 (br s,
1H), 6.00 (s, 1H), 7.10–7.18 (m, 4H). Yield 66%.
5.6.1. General procedure 1
5.6.1.1. 2-[1-(5-Ethyl-[1,3,4]thiadiazol-2-ylcarbamoyl)-
cyclopentylmethyl]-pentanoic acid tert-butyl ester, tert-
butyl-2. dH (CDCl3, 300 MHz) 0.82 (t, J = 7.0 , 3H),
1.20–1.80 (m, 22H), 1.84–1.87 (m, 1H), 2.18–2.23 (m,
4H), 3.04 (q, J = 7.5 Hz, 2H), 9.10 (br s, 1H). LRMS:
m/z (TS+) 396 (M+H). Yield 92%.
5.6.1.9.
3-{1-[2-(trans-4-Chlorophenyl)-cyclopropyl-
carbamoyl]-cyclopentyl}-2-methoxymethyl-propionic acid
tert-butyl ester, tert-butyl-20. dH (CDCl3, 400 MHz)
1.15–1.30 (m, 4H), 1.50–1.70 (m, 1H), 1.80–1.90 (m,
1H), 2.40–3.55 (m, 1H), 4.20 (dd, J = 5.7, 1.1 Hz, 2H),
6.95 (d, J = 8.8 Hz, 2H), 7.20–7.28 (m, 2H). HRMS:
m/z Found 436.2242. C24H35NO4Cl requires M+H.
436.2249, Yield 40%.
5.6.1.2.
2-[1-(5-Benzyl-[1,3,4]thiadiazol-2-ylcarba-
moyl)-cyclopentylmethyl]-4-methoxy-butyric acid tert-
butyl ester, tert-butyl-14. dH (CDCl3, 400 MHz) 1.32 (s,
9H), 1.54–2.31 (m, 13H), 3.20 (s, 3H), 3.25–3.33 (m,
2H), 4.30 (s, 2H), 7.22–7.33 (m, 5H), 10.49 (br s, 1H).
LRMS: (ES+) 474 (M+H). Yield 88%.
5.6.1.10. 2-{1-[2-(trans-4-Chlorophenyl)-cyclopropyl-
carbamoyl]-cyclopentylmethyl}-4-methoxy-butyric acid
tert-butyl ester, tert-butyl-21. dH (CDCl3, 400 MHz)
1.10–1.19 (m, 2H), 1.42 (s, 9H), 1.57–1.82 (m, 8H),
1.85–2.05 (m, 5H), 2.33–2.43 (m, 1H), 2.77–2.81 (m,
1H), 3.23 (s, 3H), 3.39 (dd, J = 5.7, 1.0 Hz, 2H), 6.05
(br s, 1H), 7.14 (d, J = 8.8 Hz, 2H), 7.28 (d,
J = 8.8 Hz, 2H). LRMS: m/z (TS+) 450 (M+H). Yield
86%.
5.6.1.3. 2-[1-(trans-2-Phenyl-cyclopropylcarbamoyl)-
cyclopentylmethyl]-pentanoic acid tert-butyl ester, tert-
butyl-15. dH (CDCl3, 400 MHz) 0.88 (t, J = 6.9 Hz, 3H),
1.16–1.20 (m, 1H), 1.20–1.58 (m, 16H), 1.63–1.66 (m,
5H), 1.90–2.14 (m, 4H), 2.23–2.25 (m, 1H), 2.90–2.93
(m, 1H), 6.00–6.06 (m, 1H), 7.16–7.22 (m, 3H),
7.24–7.27 (m, 2H). LRMS: m/z (ES+) 400 (M+H). Yield
86%.

D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
153
5.6.1.11.
4-Methoxy-2-[1-(trans-2-pentyl-cyclopro-
(br s, 1H), 7.10 (d, J = 8.6 Hz, 2H), 7.20 (d, J = 8.6 Hz,
2H). LRMS: m/z (TS+) 452 (M+H). Yield 68%.
pylcarbamoyl)-cyclopentylmethyl]-butyric acid tert-butyl
ester, tert-butyl-22. dH (CDCl3, 400 MHz) 0.40–0.60 (m,
2 H), 0.70–0.90 (m, 4H), 1.05–1.15 (m, 1H), 1.20–1.30
(m, 4H), 1.30–1.50 (m, 14H), 1.50–2.00 (m, 10H),
2.20–2.40 (m, 2H), 3.20–3.35 (m, 5H), 5.70–5.90 (br s,
1H). LRMS: m/z (TS+) 410 (M+H). Yield 71%.
5.6.1.19. 2-{1-[3-(4-Fluoro-phenyl)-propylcarbamoyl]-
cyclopentylmethyl}-4-methoxy-butyric acid tert-butyl
1
ester, tert-butyl-31. H NMR (400 MHz, CDCl3) 1.42
(s, 9H), 1.50–1.53 (m, 2H), 1.60–1.62 (m, 4H), 1.74–
1.82 (m, 5H), 1.89–1.93 (m, 3H), 2.27–2.31 (m, 1H),
2.63 (t, J = 6.7 Hz, 2H), 3.08–3.13 (m, 1H), 3.21 (s,
3H), 3.38 (t, J = 6.8 Hz, 2H), 5.88–5.90 (m, 1H), 6.91
(d, J = 8.6 Hz, 2H), 7.15 (d, J = 8.6 Hz, 2H). LRMS:
(ES+) 436 (M+H). Yield 67%.
5.6.1.12. 4-Methoxy-2-{1-[(trans-2-(4-methoxy-phen-
yl)-cyclopropylcarbamoyl)]-cyclopentylmethyl}-butyric
acid tert-butyl ester, tert-butyl-23. dH (CDCl3, 400 MHz)
1.00–1.10 (m, 2H), 1.40 (s, 9H), 1.35–1.45 (m, 2H), 1.50–
1.80 (m, 7H), 1.85–2.10 (m, 4H), 2.25–2.35 (m, 1H),
2.70–2.80 (m, 1H), 3.20 (s, 3H), 3.25–3.35 (m, 2H),
3.70 (s, 3H), 6.05 (br s, 1H), 6.70 (d, J = 8.6 Hz, 2H),
7.05 (d, J 8.6 Hz, 2H). LRMS: m/z (TS+) 446 (M+H).
Yield 38%.
5.6.1.20. 4-Methoxy-2-{1-[3-(4-methoxy-phenyl)-pro-
pylcarbamoyl]-cyclopentylmethyl}-butyric acid tert-butyl
ester, tert-butyl-32. 1H NMR (400 MHz, CDCl3) 1.42 (s,
9H), 1.60–1.65 (m, 5H), 1.84–1.86 (m, 4H), 2.08–2.15
(m, 1H), 2.28–1.33 (m, 1H), 2.66 (t, J = 6.8 Hz, 2H),
3.26 (s, 3H), 3.26–3.34 (m, 2H), 3.74 (s, 3H), 5.74 (br
s, 1H), 6.84 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 8.6 Hz,
2H). LRMS: (ESꢀ) 448 (MꢀH). Yield 85%.
5.6.1.13.
2-{1-[(trans-2-(4-Fluoro-phenyl)-cyclopro-
pylcarbamoyl)]-cyclopentylmethyl}-4-methoxy-butyric
acid tert-butyl ester, tert-butyl-24. dH (CDCl3, 400 MHz)
1.10–1.20 (m, 2H), 1.42 (s, 9H), 1.55–1.82 (m, 8H), 1.97–
2.12 (m, 5H), 2.33–2.43 (m, 1H), 2.77–2.88 (m, 1H), 3.24
(s, 3H), 3.37–3.45 (m, 2H), 6.05 (br s, 1H), 6.96 (d,
J = 8.6 Hz, 2H), 7.16–7.20 (m, 2H). LRMS: m/z (TS+)
434 (M+H). Yield 65%.
5.6.1.21. 4-Methoxy-2-{1-[2-(4-methoxy-phenoxy)-
ethylcarbamoyl]-cyclopentylmethyl}-butyric acid tert-bu-
1
tyl ester, tert-butyl-33. H NMR (400 MHz, CDCl3) dH
1.42 (s, 9H), 1.41–1.44 (m, 2H), 1.63–1.69 (m, 6H), 1.72–
1.75 (m, 2H), 1.98–2.05 (m, 3H), 2.36–2.39 (m, 1H), 3.17
(s, 3H), 3.25 (t, J = 6.8 Hz, 2H), 3.57–3.62 (m, 2H), 3.78
(s, 3H), 3.91 (t, J = 6.8 Hz, 2H), 6.10–6.12 (m, 1H), 6.71
(m, 4H). LRMS: (TS+) 450 (M+H). HRMS: Found
450.2842. C25H39NO6 requires M+H, 450.2840. Yield
60%.
5.6.1.14. 4-Methoxy-2-(1-phenethylcarbamoyl-cyclo-
pentylmethyl)-butyric acid benzyl ester, Bn-25. dH
(CDCl3, 400 MHz) 1.28–2.03 (m, 10H), 2.43–2.57 (m,
1H), 2.78 (t, J = 6.9 Hz, 2H), 3.20 (s, 3H), 3.24 (t,
J = 6.8 Hz, 2H), 3.39–3.50 (m, 2H), 5.03 (s, 2H), 5.64
(br s, 1H), 7.16–7.37 (m, 10H). Yield 65%.
5.7. General procedure 2. tert-Butyl ester deprotection
5.6.1.15. 4-Methoxy-2-{1-[2-(4-methoxy-phenyl)-eth-
ylcarbamoyl]-cyclopentylmethyl}-butyric acid tert-butyl
The requisite tert-butyl ester (1 equiv) was dissolved in a
1:1 (v/v) mixture of TFA and DCM (0.25 M total) and
the reaction mixture stirred at room temperature under
a nitrogen atmosphere overnight. The reaction mixture
was evaporated and then partitioned between DCM
and water. The organic layer was separated, washed sev-
eral times with water, dried (MgSO4), then evaporated
and the residue purified by flash chromatography on sil-
ica gel using mixtures of between 2% and 5% MeOH and
DCM to afford the desired acid. Alternatively, the tert-
butyl ester was dissolved in either Et2O, DCM or
EtOAc, and HCl gas bubbled through the solution at
room temperature for 10 min. The solution was then
purged by bubbling through N2 gas for a further
10 min and then evaporated to dryness. The residue
was purified by flash chromatography on silica gel as
above to afford the desired acids.
1
ester, tert-butyl-27. H NMR (400 MHz, CDCl3) 1.42
(s, 9H), 1.60–1.64 (m, 5H), 1.83–1.89 (m, 4H), 2.00–
2.04 (m, 1H), 2.33–2.37 (m, 1H), 2.68 (t, J = 6.9 Hz,
2H), 3.27 (s, 3H), 3.37–3.39 (m, 2H), 3.77 (s, 3H), 5.73
(br s, 2H), 6.85 (d, J = 8.6 Hz, 2H), 7.07 (d,
J = 8.6 Hz, 2H). LRMS: (ESꢀ) 448 (MꢀH). Yield 65%.
5.6.1.16. 2-(1-Heptylcarbamoyl-cyclopentylmethyl)-4-
methoxy-butyric acid tert-butyl ester, tert-butyl-28. dH
(CDCl3, 300 MHz) 0.82 (t, J = 6.7 Hz, 3H), 1.15–1.30
(m, 8H), 1.42 (s, 9H), 1.40–1.50 (m, 4H), 1.50–2.00 (m,
11H), 2.30–2.40 (m, 1H), 3.09–3.23 (m, 2H), 3.20 (s,
3H), 3.30 (t, J = 6.5 Hz, 2H), 5.70 (br s, 1H). Yield 86%.
5.6.1.17. 4-Methoxy-2-[1-(3-phenyl-propylcarbamoyl)-
cyclopentylmethyl]-butyric acid tert-butyl ester, tert-bu-
tyl-29. dH (CDCl3, 300 MHz) 1.40 (s, 9H), 1.45 (m, 2H),
1.60 (m, 8H), 1.80 (m, 4H), 2.30 (m, 1H), 2.60 (t,
J = 6.7 Hz, 2H), 3.20 (s, 3H), 3.30 (m, 4H), 3.80 (br s,
1H), 7.10 (d, J = 8.6 Hz, 3H), 7.20 (d, J = 8.6 Hz, 2H).
LRMS: m/z (ESꢀ) 418 (MꢀH). Yield 76%.
The following acids were prepared according to
5.7.1. General procedure 2
5.7.1.1. 2-[1-(5-Ethyl-[1,3,4]thiadiazol-2-ylcarbamoyl)-
cyclopentylmethyl]-pentanoic acid, 2. dH (CDCl3, 400 MHz)
0.92 (t, 3H, J = 7.0 Hz), 1.35 (t, 3H, J = 7.5 Hz), 1.25–1.80
(m, 11H), 2.20–2.50 (m, 4H), 2.95 (q, 2H, J = 7.5 Hz),
12.10 (br s, 1H); LRMS m/z (TS+) 340 (M+H); Anal.
Found C, 56.46; H, 7.46; N, 12.37%. (C16H25N3O3S)
requires C, 56.62; H, 7.44; N, 12.37%. Yield 86%.
5.6.1.18. 2-{1-[3-(4-Chloro-phenyl)-propylcarbamoyl]-
cyclopentylmethyl}-4-methoxy-butyric acid tert-butyl
ester, tert-butyl-30. dH (CDCl3, 400 MHz) 1.40 (s, 10H),
1.50–2.05 (m, 13H), 2.30–2.40 (m, 1H), 2.60 (t,
J = 6.7 Hz, 2H), 3.25 (s, 3H), 3.20–3.35 (m, 4H), 5.80

154
D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
5.7.1.2.
2-[1-(5-Benzyl-[1,3,4]thiadiazol-2-ylcarba-
6.00 (s, 1H), 7.12 (d, J = 8.6 Hz, 2H), 7.23 (d,
J = 8.6 Hz, 2H). LRMS: m/z (ESꢀ) 392 (MꢀH). Anal.
Found C, 63.91; H, 7.10; N, 3.53%. C21H28ClNO4 re-
quires C, 64.03; H, 7.16; N, 3.56%. Yield 74%.
moyl)-cyclopentylmethyl]-4-methoxy-butyric acid, 14. dH
(CDCl3, 400 MHz) 0.62–2.57 (m, 13H), 3.07 (s, 3H),
2.96–3.44 (m, 2H), 4.09 (s, 2H), 7.20 (br s, 5H). HRMS:
m/z Found 418.1796. C21H27N3O4S requires M+
418.1795. Anal. Found C, 60.63; H, 6.44; N, 10.37%.
(C21H27N3O4S) requires C, 60.41; H, 6.52; N, 10.06%.
Yield 64%.
5.7.1.9. 4-Methoxy-2-[1-(trans-2-pentyl-cyclopropyl-
carbamoyl)-cyclopentylmethyl]-butyric acid, 22. dH
(CDCl3, 400 MHz) 0.50–0.63 (m, 3H), 0.77–0.84 (m,
4H), 1.01–1.18 (m, 1H), 1.20–1.78 (m, 14H), 1.82–2.08
(m, 2H), 3.27 (s, 3H), 3.33–3.41 (m, 2H), 5.92 (s, 1H).
LRMS: m/z (ESꢀ) 352 (MꢀH). Anal. Found C, 67.66;
H, 10.04; N, 4.13%. (C20H35NO4) requires C, 67.95;
H, 9.98; N, 3.96%. Yield 47%.
5.7.1.3. 2-[1-(trans-2-phenyl-cyclopropylcarbamoyl)-
cyclopentylmethyl]-pentanoic acid, 15. dH (CDCl3,
400 MHz) 0.90 (t, 3H, J = 6.9 Hz), 1.12–2.14 (m, 17H),
2.32–2.42 (m, 1H), 2.84–2.92 (m, 1H), 6.10 (s, 1H),
7.09–7.17 (m, 3H), 7.20–7.30 (m, 2H). LRMS: m/z
(TS+) 344 (M+H). Yield 86%.
5.7.1.10. 4-Methoxy-2-{1-[(trans-2-(4-methoxy-phen-
yl)-cyclopropylcarbamoyl)]-cyclopentylmethyl}-butyric
acid, 23. dH (CDCl3, 400 MHz) 1.01–1.22 (m, 2H), 1.40–
2.22 (m, 15H), 2.42–2.57 (m, 1H), 2.73–2.82 (m, 1H),
3.23 (s, 3H), 3.27–3.44 (m, 2H), 3.72 (s, 3H), 6.12 (s,
1H), 6.78 (d, J = 8.6 Hz, 2H), 7.06 (d, J = 8.6 Hz, 2H).
LRMS: m/z (ESꢀ) 388 (MꢀH). Anal. Found C, 68.02;
H, 7.78; N, 3.69%. (C22H31NO5) requires C, 67.84; H,
8.02; N, 3.60%. Yield 44%.
5.7.1.4. 4-Methoxy-2-[1-(trans-2-phenyl-cyclopropyl-
carbamoyl)-cyclopentylmethyl]-butyric acid, 16. dH
(CDCl3, 400 MHz) 1.02–1.26 (m, 2H), 1.37–1.84 (m,
7H), 1.85–2.16 (m, 4H), 2.62 (br s, 1H), 2.80–2.93 (m,
1H), 3.29 (s, 3H, Me), 3.22–3.58 (m, 2H), 6.21 (br s,
1H), 7.03–7.34 (m, 5H). LRMS: m/z (TS+) 346
(M+H). HRMS: m/z Found 346.2011. C20H27NO4 re-
quires M+H. 346.2013. Anal. Found C, 69.51; H, 7.86;
N, 4.37%. (C20H27NO4) requires C, 69.54; H, 7.88; N,
4.05%. Yield 88%.
5.7.1.11.
2-{1-[(trans-2-(4-Fluoro-phenyl)-cyclopro-
pylcarbamoyl)]-cyclopentylmethyl}-4-methoxy-butyric
acid, 24. dH (CDCl3, 400 MHz) 1.01–1.16 (m, 2H), 1.40–
1.52 (m, 8H), 1.81–2.00 (m, 5H), 2.47–2.54 (m, 1H),
2.78–2.82 (m, 1H), 3.28 (s, 3H), 3.33–3.43 (m, 2H),
6.05 (br s, 1H), 6.85 (d, J = 8.6 Hz, 2H), 7.09 (d,
J = 8.6 Hz, 2H). LRMS: m/z (ESꢀ) 376 (MꢀH). HRMS:
m/z Found 378.2077. C21H28NO4F requires MH+
378.2075. Yield 64%.
5.7.1.5. 2-Methoxymethyl-3-[1-(trans-2-phenyl-cyclo-
propylcarbamoyl)-cyclopentyl]-propionic acid, 17. dH
(CDCl3, 400 MHz) 1.13–1.28 (m, 2H), 1.48–1.56 (m,
3H), 1.60 (br s, 3H), 1.81 (d, 1H), 2.01–2.06 (m, 4H),
2.61 (br s, 1H), 2.93 (br s, 1H), 3.30 (s, 3H), 3.42 (d,
1H, J = 9.3 Hz), 3.52 (d, 1H, J = 9.3 Hz), 6.40 (s, 1H),
7.08–7.16 (m, 3H), 7.26–7.37 (m, 2H). LRMS: m/z
(ESꢀ) 344 (MꢀH). Anal. Found C, 69.44; H, 7.80; N,
4.01%. C20H27NO4 requires C, 69.54; H, 7.88; N,
4.05%. Yield 45%.
5.7.1.12. 4-Methoxy-2-{1-[2-(4-methoxy-phenyl)-eth-
ylcarbamoyl]-cyclopentylmethyl}-butyric acid, 27. dH
(CDCl3, 400 MHz) 1.55–1.63 (m, 8H), 1.90–2.04 (m,
3H), 2.16 (dd, 1H), 2.54–2.59 (m, 1H), 2.75 (t,
J = 6.8 Hz, 2H), 3.23 (s, 3H), 3.41–3.46 (m, 2H), 3.57
(t, J = 6.8 Hz, 2H), 3.85 (s, 3H), 5.84–5.89 (m, 1H),
6.85 (d, J = 8.6 Hz, 2H), 7.13 (d, J = 8.6 Hz, 2H).
LRMS: (ESꢀ) 376 (MꢀH). Anal. Found C, 64.57; H,
8.09; N, 3.36%. C21H31NO5Æ0.6H2O requires C, 64.96;
H, 8.36; N, 3.61%. Yield 43%.
5.7.1.6. 2-[1-(2-Hydroxymethyl-indan-2-ylcarbamoyl)-
cyclopentylmethyl]-4-methoxy-butyric acid 19. dH
(CDCl3, 400 MHz) 1.43–1.76 (m, 7H), 1.80–2.24 (m,
4H), 2.57–2.68 (m, 2H), 3.06 (d, 1H J = 16.5 Hz), 3.12
(d, 1H J = 15.7 Hz), 3.27–3.31 (m, 1H), 3.32 (s, 3H),
3.36–3.48 (m, 2H), 3.80 (d, 1H J = 6.5 Hz), 3.87 (d, 1H
J = 6.5 Hz), 6.04 (s, 1H), 7.16–7.22 (m, 4H). Anal.
Found C, 70.61; H, 8.37; N, 3.74%. C22H31NO4 requires
C, 70.75; H, 8.37; N, 3.75%. Yield 89%.
5.7.1.13. 2-(1-Heptylcarbamoyl-cyclopentylmethyl)-4-
methoxy-butyric acid, 28. dH (CDCl3, 400 MHz) 0.81 (t,
J = 6.7 Hz, 3H), 1.11–1.35 (m, 8H), 1.45–1.70 (m, 10H),
1.75 (d, J = 6.5 Hz, 2H), 1.85–1.95 (m, 2H), 1.95–2.14
(m, 2H), 2.40–2.50 (m, 1H), 3.15–3.25 (m, 2H), 3.30 (s,
3H), 3.47–3.53 (m, 2H), 6.23 (br s, 1H). LRMS: m/z
(TS+) 342 (M+H+). HRMS: m/z Found 242.2656.
C19H35NO4 requires M+ 342.2639. Yield 64%.
5.7.1.7. 3-{1-[2-(trans-4-Chlorophenyl)-cyclopropylcar-
bamoyl]-cyclopentyl}-2-methoxymethyl-propionic acid, 20.
dH (CDCl3, 400 MHz) 1.04–1.18 (m, 2H), 1.20–1.36 (m,
2H), 1.36–1.79 (m, 7H), 1.83–2.08 (m, 4H), 2.57–2.66
(m, 1H), 2.73–2.83 (m, 1H), 3.27 (s, 3H), 3.38 (t, 1H
J = 9.3 Hz), 3.49 (t, 1H J = 9.3 Hz), 6.21 (br s, 1H),
7.03 (d, 2H J = 8.8 Hz), 7.18 (d, 2H J = 8.8 Hz). LRMS:
m/z (ESꢀ) 378 (MꢀH). HRMS: m/z Found 380.1622.
C20H26ClNO4 requires MH+ 380.1623. Yield 12%.
5.7.1.14. 4-Methoxy-2-[1-(3-phenyl-propylcarbamoyl)-
cyclopentylmethyl]-butyric acid, 29. dH (CDCl3,
400 MHz) 1.50–1.60 (m, 10H), 1.80–1.86 (m, 3H),
1.90–2.00 (m, 2H), 2.50–2.55 (m, 1H), 2.60 (t,
J = 6.7 Hz, 2H), 3.20–3.26 (m, 4H), 3.40 (t, J = 6.7 Hz,
2H), 5.90 (br s, 1H), 7.10–7.30 (m, 5H). LRMS: m/z
(TS+) 362 (M+H). Anal. Found C, 69.70; H, 8.65; N,
3.86%. C21H31NO4 requires C, 69.78; H, 8.64; N,
3.87%. Yield 68%.
5.7.1.8.
2-{1-[2-(trans-4-Chlorophenyl)-cyclopropyl-
carbamoyl]-cyclopentylmethyl}-4-methoxy-butyric acid,
21. dH (CDCl3, 400 MHz) 1.10–1.20 (m, 2H), 1.50–
1.70 (m, 8H), 1.85–2.05 (m, 5H), 2.55–2.65 (m, 1H),
2.72–2.85 (m, 1H), 3.20 (s, 3H), 3.31–3.43 (m, 2H),

D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
155
5.7.1.15. 2-{1-[3-(4-Chloro-phenyl)-propylcarbamoyl]-
cyclopentylmethyl}-4-methoxy-butyric acid, 30. dH
(CDCl3, 400 MHz) 1.50–1.75 (m, 9H), 1.80–1.95 (m,
5H), 2.05–2.50 (m, 2H), 2.60 (t, J = 6.7 Hz, 2H), 3.25
(s, 3H), 3.20–3.30 (m, 2H), 3.35–3.40 (m, 2H), 6.10 (br
s, 1H), 7.10 (d, J = 8.6 Hz, 2H), 7.20 (d, J = 8.6 Hz,
2H). LRMS: m/z (TS+) 396 (M+H). HRMS: m/z Found
396.1943. C21H31ClNO4 requires M+ 396.1936. Anal.
Found C, 63.73: H, 7.84; N, 3.61%. C21H30NO4Cl re-
quires C, 63.71; H, 7.64; N, 3.54%. Yield 84%.
5.8.1.2.
4-Methoxy-2-(1-phenethylcarbamoyl-cyclo-
pentylmethyl)-butyric acid, 25. dH (CDCl3, 400 MHz)
1.42–2.08 (m, 12H), 2.43–2.57 (m, 1H), 2.83 (t,
J = 6.7 Hz, 2H), 3.30 (s, 3H), 3.28–3.43 (m, 2H), 3.47–
3.58 (m, 2H), 5.78 (br s, 1H), 7.17–7.38 (m, 5H). LRMS:
m/z (ESꢀ) 346 (MꢀH). Anal. Found C, 69.06; H, 8.42;
N, 4.01%. C20H29NO4 requires C, 69.14; H, 8.41; N,
4.03%. Yield 68%.
5.9. 1-(2-tert-Butoxycarbonyl-ethyl)-cyclopentanecarb-
oxylic acid, 3a (PG = tBu, X = H)
5.7.1.16. 2-{1-[3-(4-Fluoro-phenyl)-propylcarbamoyl]-
cyclopentylmethyl}-4-methoxy-butyric acid, 31. 1H
NMR (400 MHz, CDCl3) 1.47–1.53 (m, 2H), 1.60–1.66
(m, 4H), 1.84 (t, J = 6.7 Hz, 2H), 1.90–1.94 (m, 2H),
2.00–2.03 (m, 2H), 2.40–2.43 (m, 1H), 3.24 (s, 4H),
3.38 (t, J = 6.7 Hz, 2H), 6.08 (br s, 1H), 6.91 (d,
J = 8.6 Hz, 2H), 7.12 (d, J = 8.6 Hz, 2H), 10.45 (br s,
1H). LRMS: (ESꢀ) 379 (MꢀH). Anal. Found C,
66.35; H, 7.96; N, 3.68%. C21H30FNO4 requires C,
66.47; H, 7.97; N, 3.69%. Yield 69%.
A 2.5 M solution of nBuLi in hexanes (42 ml, 105 mmol)
was added dropwise to a stirred solution of diisopropyl-
amine (15 ml, 107 mmol) in 100 ml THF at ꢀ20 ꢁC un-
der nitrogen, and the whole stirred at this temperature
for 30 min. A solution of cyclopentane carboxylic acid
(5.7 g, 50 mmol) in 10 ml dry THF was then added
dropwise and the resulting solution stirred at ꢀ20 ꢁC
for 30 min and then at room temperature for 2 h. The
solution was then recooled to ꢀ20 ꢁC and tert-butyl
bromopropionate (11 g, 53 mmol) was added portion-
wise and the mixture then stirred at room temperature
for approximately 2 h. Hundred and fifty millilitres of
diethyl ether and 110 ml of 2 N HCl solution were then
added in sequence, and the organic layer was separated,
dried over MgSO4 and evaporated to a yellow oil. This
oil was taken up in diethyl ether (100 ml) and washed
with saturated NaHCO3 until almost all of the starting
cyclopentane carboxylic acid was no longer present by
TLC. The organics were then washed with 2 N HCl
solution, separated, dried and evaporated in vacuo to
provide 4.5 g of a yellow oil which slowly solidified on
standing. This solid was recrystallised from approxi-
mately 15 ml of hexane to provide 3.49 g of the title
5.7.1.17. 4-Methoxy-2-{1-[3-(4-methoxy-phenyl)-pro-
pylcarbamoyl]-cyclopentylmethyl}-butyric acid, 32. 1H
NMR (400 MHz, CDCl3) 1.41–1.55 (m, 2H), 1.66–1.75
(m, 6H), 1.75–1.85 (m, 2H), 2.41–2.51 (m, 1H), 2.64 (t,
J = 6.7 Hz, 2H), 3.23 (s, 3H), 3.27–3.38 (m, 2H), 3.46
(t, J = 6.7 Hz, 2H), 3.88 (s, 3H), 5.99 (br s, 1H), 6.89
(d, J = 8.6 Hz, 2H), 7.16 (d, J = 8.6 Hz, 2H). LRMS:
(ESꢀ) 390 (MꢀH). HRMS: Found 392.2430. M+H. re-
quires C22H33NO5 392.2432. Yield 84%.
5.7.1.18.
4-Methoxy-2-{1-[2-(4-methoxy-phenoxy)-
1
ethylcarbamoyl]-cyclopentylmethyl}-butyric acid, 33. H
NMR (400 MHz, CDCl3) dH 1.63–1.68 (m, 4H), 2.02–
2.22 (m, 2H), 2.12–2.18 (m, 2H), 2.50–2.56 (m, 1H),
3.22 (s, 3H), 3.37 (t, J = 6.7 Hz, 2H), 3.68 (t,
J = 6.7 Hz, 2H), 3.76 (s, 3H), 4.04 (t, J = 6.7 Hz, 2H),
6.24 (br s, 1H), 6.80–6.92 (m, 4H). LRMS: (ESꢀ) 392
(MꢀH). HRMS: Found, 394.2222. C21H32NO6 requires
M+H, 394.2225. Yield 87%.
compound as a white solid, 29%. dH (CDCl3,
400 MHz) 1.40 (s, 9H), 1.42–1.78 (m, 6H), 1.84–1.95
(m, 2H), 2.03–2.42 (m, 4H).
5.10. General procedure 4. Preparation of acids 3
5.10.1. 1-(2-tert-Butoxycarbonyl-pent-4-enyl)-cyclopen-
tanecarboxylic acid, 3b (PG = tBu, X = allyl); 1-(2-tert-
butoxycarbonyl-pentyl)-cyclopentanecarboxylic acid, 3c
(PG = tBu, X = n-propyl); 1-(2-tert-butoxycarbonyl-3-
5.8. General procedure 3. Benzyl ester deprotection
The benzyl ester (1 equiv) was dissolved in EtOH (1 M),
10 wt % Pd/C was added and the whole was shaken un-
der a 15 psi hydrogen atmosphere for 3 h. The mixture
was passed through a short plug of Arbocel to remove
the catalyst and filtrate evaporated to dryness to afford
the desired acids which were found to be of sufficient
purity to be used with no further purification.
methoxy-propyl)-cyclopentanecarboxylic
acid,
3d
(PG = tBu, X = methoxymethyl); 1-(2-tert-butoxycarbon-
yl-4-methoxy-butyl)-cyclopentanecarboxylic acid, 3e
(PG = tBu, X = methoxyethyl). 1-(2-tert-Butoxycarbon-
yl-ethyl)-cyclopentanecarboxylic acid3,7 (3a, 1 equiv)
was added to a stirred solution of lithium diisopropyla-
mide (2.2 equiv) in dry THF (0.27 M total concentra-
tion) at ꢀ78 ꢁC under nitrogen and the mixture stirred
at this temperature for 45 min. A solution of the electro-
phile (1.1 equiv, allyl bromide, chloromethyl methyl
ether or bromoethyl methyl ether) in dry THF was add-
ed dropwise over 30 min and stirring continued for a
further 30 min before the reaction mixture was allowed
to slowly warm to room temperature overnight. The
bright orange reaction mixture was cooled to 0 ꢁC and
quenched with 2 N HCl. The solvent was removed in
vacuo and the mixture extracted with EtOAc. The com-
bined extracts were washed with brine, dried (MgSO4)
The following acids were prepared according to
5.8.1. General procedure 3
5.8.1.1.
2-[1-(4-Butyl-pyridin-2-ylcarbamoyl)-cyclo-
pentylmethyl]-4-methoxy-butyric acid, 18. dH (CDCl3,
400 MHz) 0.92 (t, 3H, Me J = 6.9 Hz), 1.24–2.60 (m,
19H), 3.25 (s, 3H), 3.39 (t, 2H, J = 7.3 Hz), 6.68–6.71
(m, 1H), 7.63–7.70 (m, 1H), 8.21 (s, 1H), 9.77 (br s,
1H). LRMS: m/z (ESꢀ) 376 (MꢀH). Anal. Found C,
66.20; H, 8.60; N, 7.35%. C21H32N2O4Æ0.3H2O requires
C, 66.05; H, 8.60; N, 7.34%. Yield 97%.

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D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
and evaporated to an orange oil which was purified by
flash chromatography (SiO2; 5–10% EtOAc in pentane).
The products7 3b, 3d and 3e were isolated as pale yellow
oils. Compound 3b was taken up in EtOH at room tem-
perature and 10 wt % Pd/C added in one portion. The
mixture was shaken under a hydrogen atmosphere of
15 psi for 1 h and then filtered to remove the catalyst.
The filtrate was evaporated in vacuo to a clear oil of
3c which was used with no further purification.
100 ml) to remove any unreacted starting material and
the aqueous layer was then acidified to pH 1 with 2 N
HCl solution. This was then extracted with ether (2·
100 ml), dried over MgSO4 and evaporated in vacuo
to give the title compound as a light brown oil (39 g).
This oil was taken up in piperidine (2.6 ml, 0.026 mol)
and dry pyridine (300 ml), and paraformaldehyde
(7.1 g, 0.24 mol) added in one portion. The mixture
was stirred at 60 ꢁC for 3 h and then cooled to room
temperature. The reaction mixture was poured onto
ice, acidified to pH 1 with 2 N HCl and extracted with
EtOAc (2· 100 ml). The combined organics were then
washed with water (40 ml), saturated aq NaHCO3 solu-
tion (40 ml) and then water (40 ml), and then dried over
MgSO4. The dried solution was then filtered and evapo-
rated in vacuo to a clear oil. This oil was purified using
silica gel chromatography and 4/1 hexane/ether as eluant
to provide the title compound as a clear oil (17g, 46%).
dH (CDCl3, 400 MHz) 2.44 (t, J = 7.6 Hz, 2H), 3.46 (s,
3H), 3.62 (t, J = 7.6 Hz, 2H), 5.14 (s, 2H), 5.57 (s,
1H), 6.22 (s, 1H), 7.25–7.39 (m, 5H).
Compound 3b; PG = tBu, X = allyl; dH (CDCl3,
400 MHz) 1.44 (s, 9H), 1.48–1.83 (m, 6H), 2.02–2.08
(m, 2H), 2.16–2.32 (m, 3H), 2.51–2.59 (m, 1H), 2.63–
2.72 (m, 1H), 4.99 (dd, 1H), 5.17 (dd, 1H), 5.76 (dddd,
1H); Anal. Found C, 68.35; H, 9.26; N, 0%. C16H26O4
requires C, 68.06; H, 9.28; N, 0%, Yield 92%. 3c;
PG = tBu, X = n-propyl; dH (CDCl3, 300 MHz) 0.86 (t,
3H), 1.22–1.58 (m, 15H), 1.64 (m, 4H), 1.78 (dd. 1H),
2.00–2.18 (m, 3H), 2.24 (m, 1H). LRMS: m/z (TSꢀ)
283 (MꢀH), Yield 91%. 3d; PG = tBu, X = methoxym-
ethyl; dH (CDCl3, 300 MHz) 1.40 (s, 9H), 1.40–1.50
(m, 4H), 1.20–1.80 (m, 1H), 1.80–1.90 (m, 1H), 2.00
(dd, 1H), 2.00–2.05 (m, 3H), 2.20 (dd, 1H), 2.50–2.60
(m, 1H), 3.30 (s, 1H), 3.30–3.40 (m, 1H), 3.40 (t, 1H).
LRMS: m/z (TS+) 304 ðMNHþ4 Þ, Yield 66%. 3e;
PG = tBu, X = 2-methoxyethyl; dH (CDCl3, 400 MHz)
1.40 (s, 9H), 1.40–1.70 (m, 7H), 1.75–1.95 (m, 2H),
2.00–2.15 (m, 3H), 2.30–2.40 (m, 1H), 3.30 (s, 3H),
3.30–3.40 (m, 2H). LRMS: m/z (ESꢀ) 299 (MꢀH), Yield
55%.
5.10.2.3. Step 3. 1-(2-Benzyloxycarbonyl-4-meth-
oxybutyl)cyclopentane carboxylic acid, 3f. nBuLi
(61.7 ml, 0.15 mol, 2.5 M solution in hexanes) was add-
ed dropwise via syringe to a stirred solution of diisopro-
pylamine (21.4 ml, 0.15 mol) in dry THF (60 ml) under a
nitrogen atmosphere at ꢀ30 ꢁC. Once the addition was
complete, the mixture was stirred at this temperature
for 30 min, before adding a solution of cyclopentane
carboxylic acid (8.8 g, 0.08 mol) in 30 ml dry THF.
The reaction mixture was allowed to warm to room tem-
perature over 90 min, and stirring was then continued at
room temperature for a further 1 h, before cooling to
ꢀ78 ꢁC. The product from the above step (17 g,
0.08 mol) in 30 ml dry THF was added dropwise, and
the mixture stirred at ꢀ78 ꢁC for 2 h. The reaction mix-
ture was allowed to warm to 0 ꢁC over 2 h, and then
quenched by the addition of 2 N HCl to approximately
pH 2, and the reaction mixture then extracted with hex-
ane (2· 100 ml). The combined organics were washed
with water (40 ml), saturated aq NaHCO3 solution (4·
40 ml) and were then dried over MgSO4. Filtration
and evaporation in vacuo provided a light brown oil.
This oil was purified by silica gel chromatography using
a gradient of hexane and EtOAc (70/30 ! 50/50 ! 20/
80) to provide the title compound as a clear oil (8.8 g,
34%). dH (CDCl3, 400 MHz) 1.44–2.26 (m, 14H), 2.64–
2.70 (M, 1H), 3.29 (s, 3H), 3.42 (t, J = 6.7 Hz, 2H),
5.10 (s, 2H), 7.04–7.23 (m, 5H).
5.10.2. 1-(Benzyloxycarbonyl-4-methoxy-butyl)-cyclopen-
tanecarboxylic acid, 3f (PG = Bn, X = methoxyethyl)
5.10.2.1. Step 1. 2-(2-Methoxyethyl)malonic acid
dibenzyl ester. Dibenzylmalonate (71.1 g, 0.25 mol) was
added dropwise over approximately 1 h to a stirred sus-
pension of sodium hydride (7.9 g, 0.26 mol, 80% disper-
sion in mineral oil) in dry THF (250 ml) under a
nitrogen atmosphere at room temperature. During the
addition, the temperature was allowed to rise to 40 ꢁC.
A
solution of bromoethyl methyl ether (34.8 g,
0.25 mol) in THF (10 ml) was then added to the mixture
at between 30 and 40 ꢁC and the whole then stirred at
reflux for 16h. The reaction mixture was cooled to room
temperature and diluted with water (40 ml) and brine
(40 ml) and then extracted with DCM (2· 200 ml).
The combined extracts were washed with water
(40 ml), dried over MgSO4 and then evaporated to pro-
vide a yellow oil. This oil was purified by silica gel chro-
matography using 20% ether in hexane as eluant to
provide the title compound as a clear oil (58 g, 70%).
dH (CDCl3, 400 MHz) 2.01 (q, J = 7.2 Hz, 2H), 3.31 (s,
3H), 3.48 (t, J = 7.2 Hz, 1H), 3.55–3.59 (m, 2H), 5.10
(s, 4H), 7.16–7.26 (m, 10H).
5.11. General procedure 5. Preparation of cyclopropyl-
amines 7
5.11.1. trans-2-(4-Chlorophenyl)-cyclopropylamine, 7a
(R = Cl); trans-2-(4-methoxyphenyl)-cyclopropylamine,
7b (R = OMe); trans-2-(4-fluorophenyl)-cyclopropyl-
amine, 7c (R = F)
5.11.1.1. Step 1. Cyclopropanation. A mixture of the
requisite styrene 4 (1 equiv) and rhodium acetate dimer
(0.05 equiv) in toluene (2 M) was heated to 85 ꢁC before
the addition of ethyl diazoacetate (1 equiv) over 30 min
and the whole was then heated at 80 ꢁC for a further 1 h.
5.10.2.2. Step 2. 4-Methoxy-2-methylene-butyric acid
benzyl ester. The product from the above step (58 g,
0.17 mol) was taken up in dioxan (350 ml) at room tem-
perature and a solution of KOH (10 g) in 150 ml water
was added dropwise. The whole was stirred at room
temperature for 16 h, and then evaporated in vacuo to
approximately 100 ml volume and then 200 ml of water
added. This solution was extracted with ether (3·

D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
157
The solution was concentrated in vacuo, and the residue
purified by flash column chromatography using DCM
as eluant to provide the cyclopropane carboxylate esters
5 as oils.
5.11.6. trans-2-(4-Methoxyphenyl)-cyclopropane carbox-
ylic acid, 6b; R = OMe. dH (CDCl3, 400 MHz) 1.30–1.41
(m, 1H), 1.58–1.69 (m, 1H), 1.79–1.90 (m, 1H), 2.53–
2.62 (m, 1H), 6.83 (d, J = 8.4 Hz, 2H), 7.04 (d,
J = 8.4 Hz, 2H). Yield 78%.
The following cyclopropanes were prepared according
to Step 1;
5.11.7. trans-2-(4-Fluorophenyl)-cyclopropane carboxylic
acid, 6c: R = F. dH (CDCl3, 400 MHz) 1.40–1.46 (m,
1H), 1.78–1.85 (m, 1H), 2.57 (dd, J = 16.0, 7.8 Hz,
1H), 2.83 (dd, J = 16.0, 7.8 Hz, 1H), 6.77 (d,
J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H). Yield 78%.
5.11.2. trans-2-(4-Chlorophenyl)-cyclopropane carboxylic
acid ethyl ester, 5a; R = Cl. dH (CDCl3, 400 MHz) 1.15–
1.30 (m, 4H), 1.50–1.70 (m, 1H), 1.80–1.90 (m, 1H),
2.40–3.55 (m, 1H), 4.20 (q, J = 7.2 Hz, 2H), 6.95 (d,
J = 8.6 Hz, 2H), 7.24 (d, J = 8.6 Hz, 2H). LRMS: m/z
(TS+) 242 (M+NH4þ). Yield 37%.
5.11.7.1. Step 3. Curtius rearrangement. The products
6 from Step 2 (1 equiv), DPPA (1.1 equiv) and triethyl-
amine (1.5 equiv) were combined in tert-butanol (0.5 M)
under nitrogen and heated at 90 ꢁC for 48 h before cool-
ing to room temperature. The mixture was diluted with
EtOAc (20 ml) and saturated Na2CO3 solution (20 ml)
and the organic layer separated. The aqueous layer
was re-extracted with EtOAc (20 ml) and the combined
organic layers were dried over MgSO4, filtered and evap-
orated. The resulting residue was purified by flash chro-
matography using 20% EtOAc in pentane as eluant to
provide the intermediate tert-butyl carbamates as white
solids. The carbamates were then taken up in EtOAc
(0.2 M), cooled to 0 ꢁC and hydrogen chloride gas was
bubbled through the solution for 30 min. The solution
was then concentrated in vacuo to give a pale yellow sol-
id of the amine HCl salt 7.
5.11.3. trans-2-(4-Methoxyphenyl)-cyclopropane carbox-
ylic acid ethyl ester, 5b; R = OMe. dH (CDCl3,
400 MHz) 1.20–1.38 (m, 5H), 1.83 (dd, J = 16.0,
7.8 Hz, 1H), 2.50 (dd, J = 16.0, 9.3 Hz, 1H), 3.80 (s,
3H), 4.16 (q, J = 7.2 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H),
7.03 (d, J = 8.6 Hz, 2H). Yield 31%.
5.11.4. trans-2-(4-Fluorophenyl)-cyclopropane carboxylic
acid ethyl ester, 5c; R = F. dH (CDCl3, 400 MHz) 1.07 (t,
J = 7.2 Hz, 3H), 1.30–1.39 (m, 1H), 1.74–1.78 (m, 1H),
2.13 (dd, J = 16.0, 7.8 Hz, 1H), 2.45 (dd, J = 16.0,
7.8 Hz, 1H), 3.86 (q, J = 7.2 Hz, 2H), 6.82 (d,
J = 8.4 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H). Yield 41%.
5.11.4.1. Step 2. Formation of trans-cyclopropane
carboxylic acids. The carboxylate esters 5 from Step 1
(1 equiv) were dissolved in ethanol (2 M) at room tem-
perature under a nitrogen atmosphere, and sodium
methoxide (4 equiv) was added portionwise over
15 min. After the addition was complete the combined
mixture was refluxed for 18h. The solution was concen-
trated in vacuo and the resulting residue diluted with
DCM (100 ml) and water (50 ml). The organic layer
was removed, and the aqueous layer re-extracted with
DCM (2·50 ml). The combined organics were dried
over MgSO4 and then evaporated to provide a clear
oil. Acidification of the aqueous layers with concd
HCl to pH 1 resulted in a white precipitate, which was
filtered and dried under vacuum to provide a white pow-
der. The oil was taken up in methanol (50 ml) and water
(50 ml) and solid LiOH (1 equiv) added. The resulting
clear solution was then heated at 70 ꢁC overnight. The
reaction mixture was cooled, concentrated in vacuo
and acidified with concd HCl to pH 1. The resulting
white precipitate was extracted with EtOAc (3· 50 ml),
and the combined organic extracts were dried with
MgSO4 and then evaporated to dryness and combined
with the white solid from above, to provide the trans
acids 6.
The following cyclopropanes were prepared according
to Step 3;
5.11.8. trans-2-(4-Chlorophenyl)-cyclopropylamine, 7a;
R = Cl. dH (CDCl3, 400 MHz) 1.30–1.40 (m, 1H),
1.40–1.50 (m, 1H), 2.30–2.40 (m, 1H), 2.80–2.90 (m,
1H), 7.15 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 8.6Hz, 2H).
Yield 81%.
5.11.9. trans-2-(4-Methoxyphenyl)-cyclopropylamine, 7b;
R = OMe. dH (CDCl3, 400 MHz) 0.87–1.04 (m, 2H),
1.79–1.90 (m, 1H), 2.43–2.54 (m, 1H), 3.80 (s, 3H),
6.80 (d, J = 8.6Hz, 2H), 6.98 (d, J = 8.6Hz, 2H). Yield
89%.
5.11.10. trans-2-(4-Fluorophenyl)-cyclopropylamine, 7c;
R = F. dH (CDCl3, 400 MHz) 0.93–0.97 (m, 2H), 2.03
(dd, J = 16.0, 7.8 Hz, 1H), 2.05 (dd, J = 16.0, 7.8 Hz,
1H), 6.86 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 8.6 Hz, 2H).
Yield 80%.
5.12. General procedure 6. Preparation of phenpropyl-
amines 9
5.12.1. 3-(4-Methoxy-phenyl)-propylamine, 9a (R = OMe);
3-(4-fluoro-phenyl)-propylamine, 9b (R = F). A solution
of the halide 8 (1 equiv), acrylonitrile (1.1 equiv), tri-o-
tolylphosphine (0.1 equiv), palladium acetate (0.1 equiv)
and triethylamine (3 equiv) in acetonitrile (0.2 M) was
refluxed under nitrogen for 14 h. The reaction mixture
was diluted with EtOAc (50 ml), and washed with 2 M
NaHCO3 (100 ml), and the organic layer was dried over
MgSO4 and filtered. The filtrate was evaporated in
The following cyclopropanes were prepared according
to Step 2;
5.11.5. trans-2-(4-Chlorophenyl)-cyclopropane carboxylic
acid, 6a; R = Cl. dH (CDCl3, 400 MHz) 1.30–1.40 (m,
1H), 1.60–1.70 (m, 1H), 1.80–1.90 (m, 1H), 2.50–2.60
(m, 1H), 7.00 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4 Hz,
2H). LRMS: m/z (ESꢀ) 195 (MꢀH). Yield 96%.

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D. C. Pryde et al. / Bioorg. Med. Chem. 15 (2007) 142–159
vacuo and the residue purified by column chromatog-
raphy using 5% EtOAc in pentane as eluant to provide
the diastereomeric vinyl nitriles, usually favouring the
trans isomer. The products from this step (1 equiv)
were taken up in ammonium hydroxide solution and
ethanol (1:1 mixture, to ca. 0.15 M) and the mixture
was shaken under 40 psi hydrogen pressure with Raney
Ni (20 wt %) for 12 h. The reaction mixture was filtered
through Arbocel and washed with ethanol (20 ml). The
filtrate was evaporated in vacuo to give the amines 9
which were pure enough to be used with no further
purification.
which was purified by column chromatography using
0%, then 1, then 2, then 5% methanol in DCM to pro-
vide the ring opened phenethylamine 26 as a clear oil
1
(319 mg, 90%). H NMR (400 MHz, CDCl3) dH 1.12
(d, 3H J = 6.5 Hz), 1.53–2.00 (m, 13H), 2.47–2.52 (m,
1H), 2.78–2.83 (m, 2H), 3.31–3.38 (m, 3H), 3.42–3.46
(m, 2H), 4.20–4.23 (m, 1H), 5.58–5.63 (m, 1H), 7.10–
7.15 (m, 3H), 7.20–7.24 (m, 2H). LRMS: (ESꢀ) 362
(MꢀH). Anal. Found C, 65.87; H, 8.40; N, 3.61%.
C21H29NO4Æ0.3DCM requires C, 66.11; H, 8.23; N,
3.62%.
5.15. General procedure 7. Classical resolution of acids 3
The following amines were prepared according to
The racemic acid 3 (1 equiv) and (+)-pseudoephedrine
(1 equiv) were dissolved in hexane (which may require
heating with a heat gun). The solution was allowed to
cool to room temperature overnight, by which time
white crystals had appeared. These were filtered off
and re-subjected to the crystallisation procedure in
the minimum quantity of hexane. Typically 9–10
recrystallisations were required to obtain material of
>90% ee. Optical purity at this stage could be estimat-
5.12.2. General procedure 6
5.12.2.1. 3-(4-Methoxy-phenyl)-propylamine, 9a;
1
R = OMe. H NMR (400 MHz, CDCl3) 1.70–1.76 (m,
2H), 2.06 (br s, 2H), 2.52 (t, J = 7.4Hz, 2H), 2.68–2.74
(m, 2H), 3.78 (s, 3H), 6.78 (d, 2H), 7.03 (d, 2H). LRMS:
(TS+) 376 (M+H). Yield 43%.
5.12.2.2. 3-(4-Fluoro-phenyl)-propylamine, 9b; R = F.
1H NMR (400 MHz, CDCl3) 1.70–1.78 (m, 2H), 2.25 (br
s, 2H), 2.47–2.50 (t, 2H), 2.64 (br s, 2H), 6.97 (d,
J = 8.6Hz, 2H), 7.20 (d, J = 8.6Hz, 2H). LRMS: (TS+)
364 (M+H). Yield 88%.
1
ed from the H NMR shifts of the diastereomeric pseu-
doephedrine salts in CDCl3—the tert-butyl ester peaks
typically showed some separation, and the relative
abundance of each peak gave a good estimate of enan-
tiomeric excess. The salt was then taken up in EtOAc,
washed with 0.5 M HCl (aq) and the organic layer
dried over MgSO4, filtered and evaporated in vacuo
to provide the R-alkyl versions of 3b–c, and the S-alk-
oxyethyl versions of 3d–e, in typically 30% yield. These
acids were then coupled to the requisite amine accord-
ing to Scheme 1. Examples of non-racemic material
synthesised according to
5.13. 2-(4-Methoxy-phenoxy)-ethyl amine, 13
Triphenylphosphine (1.78 g, 6.8 mmol) was dissolved in
20 ml DCM at room temperature under nitrogen and
first DEAD (1.18 ml, 6.8 mmol), then ethyl hydroxy
phthalimide (1 g, 5.2 mmol) were added portionwise.
The orange solution was stirred for 4 h, and the solvents
then removed under vacuum. The resulting orange resi-
due was purified by column chromatography using
DCM as eluant to provide the phenoxy compound as
an orange solid (768 mg, 50%). This was taken up in
methanol (5 ml), hydrazine hydrate added (150 ll,
3.1 mmol) in one portion and the whole refluxed for
4 h. A white precipitate was formed upon cooling, and
the suspension was stirred at room temperature for
48 h. The methanol was removed in vacuo, and the res-
idue partitioned between EtOAc (50 ml) and citric acid
(50 ml), and the layers separated. The aqueous layer
was basified to pH 8 with 1 N NaOH and extracted with
EtOAc (2· 20 ml). The organic layers were combined,
dried (MgSO4) and evaporated to give a colourless oil
of the title amine 13 (270 mg, 63%). 1H NMR
(400 MHz, CDCl3) dH 1.72 (br s, 2H), 3.02 (t,
J = 7.2 Hz, 2H), 3.76 (s, 3H), 3.95 (t, J = 7.2 Hz, 2H),
6.80–6.90 (m, 4H). LRMS: m/z (TS+) 168 (M+H).
5.15.1. General procedure 7
5.15.1.1. R-2, R-2-[1-(5-Ethyl-[1,3,4]thiadiazol-2-yl-
carbamoyl)-cyclopentylmethyl]-pentanoic acid. [a]D ꢀ9.0
(c 0.1, MeOH), Anal. Found C, 56.46; H, 7.46; N,
12.37%. (C16H25N3O3S) requires C, 56.62; H, 7.44; N,
12.37%.
5.15.1.2. S-30, 2-{1-[3-(4-Chloro-phenyl)-propylcarba-
moyl]-cyclopentylmethyl}-4-methoxy-butyric acid. [a]D
(for Na salt) ꢀ0.06 (c 3.2, MeOH), Anal. Found C,
63.73: H, 7.84; N, 3.61%. C21H30NO4Cl requires C,
63.71; H, 7.64; N, 3.54%.
5.15.1.3. S-31, 2-{1-[3-(4-Fluoro-phenyl)-propylcarba-
moyl]-cyclopentylmethyl}-4-methoxy-butyric acid. [a]D
ꢀ0.01 (c 1.87, EtOH), HRMS: m/z Found 380.2232;
C21H31NO4F requires MH+ 380.2225; Anal. Found C,
66.73: H, 8.04; N, 4.11%. C21H30NO4F requires C,
66.47; H, 7.97; N, 3.69%.
5.14. 4-Methoxy-2-[1-(1-methyl-2-phenyl-ethylcarba-
moyl)-cyclopentylmethyl]-butyric acid, 26
The benzyl ester 10 (473 mg, 1.18 mmol) was taken up in
ethanol (10 ml) at room temperature and hydrogenated
at 60 psi hydrogen pressure over 10 wt % Pd/C for 16 h.
The reaction mixture was filtered through a short plug
of Arbocel and washed with ethanol. The combined fil-
trates were evaporated in vacuo to give a colourless oil,
References and notes
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12. A fuller exploration of the in vitro and in vivo metabolism
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13. Jeng, A. Y.; Ansell, J.; Erion, M. D. Life Sci. 1989, 45,
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