Ligands to the (IRAP)/AT4 receptor encompassing a 4-hydroxydiphenylmethane scaffold replacing Tyr2

Article abstract of DOI:10.1016/j.bmc.2008.05.046

Analogues of the hexapeptide angiotensin IV (Ang IV, Val¹-Tyr²-Ile³-His⁴-Pro⁵-Phe⁶) encompassing a 4-hydroxydiphenylmethane scaffold replacing Tyr² and a phenylacetic or benzoic acid moiety replacing His⁴-Pro⁵-Phe⁶ have been synthesized and evaluated in biological assays. The analogues inhibited the proteolytic activity of cystinyl aminopeptidase (CAP), frequently referred to as the insulin-regulated aminopeptidase (IRAP), and were found less efficient as inhibitors of aminopeptidase N (AP-N). The best Ang IV mimetics in the series were approximately 20 times less potent than Ang IV as IRAP inhibitors. Furthermore, it was found that the ligands at best exhibited a 140 times lower binding affinity to the membrane-bound IRAP/AT4 receptor than Ang IV. Although the best compounds still exert lower activities than Ang IV, it is notable that these compounds comprise only two amino acid residues and are considerably less peptidic in character than the majority of the Ang IV analogues previously reported as IRAP inhibitors in the literature.

Full text of DOI:10.1016/j.bmc.2008.05.046

Bioorganic & Medicinal Chemistry 16 (2008) 6924–6935
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ligands to the (IRAP)/AT4 receptor encompassing
a 4-hydroxydiphenylmethane scaffold replacing Tyr2
Hanna Andersson a, Heidi Demaegdt b, Georges Vauquelin b, Gunnar Lindeberg a,
Anders Karlén a, Mathias Hallberg c,
*
a Department of Medicinal Chemistry, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden
b Department of Molecular and Biochemical Pharmacology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium
c Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Uppsala University, PO Box 591, SE-751 24 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Analogues of the hexapeptide angiotensin IV (Ang IV, Val1-Tyr2-Ile3-His4-Pro5-Phe6) encompassing a
4-hydroxydiphenylmethane scaffold replacing Tyr2 and a phenylacetic or benzoic acid moiety replacing
His4-Pro5-Phe6 have been synthesized and evaluated in biological assays. The analogues inhibited the
proteolytic activity of cystinyl aminopeptidase (CAP), frequently referred to as the insulin-regulated ami-
nopeptidase (IRAP), and were found less efficient as inhibitors of aminopeptidase N (AP-N). The best Ang
IV mimetics in the series were approximately 20 times less potent than Ang IV as IRAP inhibitors. Fur-
thermore, it was found that the ligands at best exhibited a 140 times lower binding affinity to the mem-
brane-bound IRAP/AT4 receptor than Ang IV. Although the best compounds still exert lower activities
than Ang IV, it is notable that these compounds comprise only two amino acid residues and are consi-
derably less peptidic in character than the majority of the Ang IV analogues previously reported as IRAP
inhibitors in the literature.
Article history:
Received 22 January 2008
Revised 9 May 2008
Accepted 22 May 2008
Available online 27 May 2008
Keywords:
Angiotensin IV
Insulin-regulated aminopeptidase (IRAP)
Cystinyl aminopeptidase (CAP)
Aminopeptidase N (AP-N)
Structure–activity relationship
Peptide synthesis
Ó 2008 Elsevier Ltd. All rights reserved.
Peptide mimetic
4-Hydroxydiphenylmethane
Tyrosine mimetic
1. Introduction
in neocortex, cerebellum, dentate gyrus and CA1–CA3 subfields
of the hippocampus.19–22 These structures are all representing
Proteolytic degradation of neuropeptides often provides prod-
ucts that are essentially inactive. However, there are also
numerous examples disclosed where the fragments formed
demonstrate pronounced biological effects and outcomes that
frequently differ considerably from those exerted by the parent
peptide.1,2 Angiotensin IV (Ang IV, Val1-Tyr2-Ile3-His4-Pro5-Phe6),
a component in the renin–angiotensin system, provides a good
example of a degradation product that at least partly is exhibiting
different effects than its precursors.3–5 The angiotensin IV receptor,
first described as the specific high-affinity binding site for Ang IV,6–8
has been identified as a membrane bound enzyme of the M1
important brain areas believed to be involved with cognitive
processes. A precise mechanism explaining the observation that
Ang IV acts as a potent cognitive enhancer has not yet been pre-
sented, but some hypotheses have emerged. The fact that IRAP is
abundantly occurring in vesicles containing the insulin-sensitive
glucose transporter GLUT4,10,23 led to the hypothesis that Ang IV
is able to enhance memory and learning by modulating the
translocation of GLUT4 to the cell surface and consequently in-
crease glucose uptake in neurons.3 Alternatively, since IRAP/
AT4 receptor ligands have been shown to act by binding to IRAP
and thereby inhibiting the catalytic activity of the enzyme,24,25 it
has been proposed that Ang IV at least partly exerts its action by
prolonging the half-life of one or more of its neuropeptide sub-
strates, such as somatostatin or vasopressin that are recognized
for their ability to facilitate learning and memory.4,10,26 It has
also been suggested that alternative targets of Ang IV exist and
that IRAP may act as a classical receptor.4,25,27 Aminopeptidase
N (AP-N) constitutes one potential target candidate because the
catalytic activity was found to be sensitive to Ang IV.28 Notably,
it was recently shown that Ang IV was able to bind with high
affinity to the CAP apo-enzyme, but neither to the native CAP,
metallopeptidase family denoted cystinyl aminopeptidase (CAP),9
a
peptidase that is more often referred to as the insulin-regulated
membrane aminopeptidase (IRAP; EC 3.4.11.3).9,10
The ability of Ang IV to facilitate learning and memory in
behavioural models has attracted a particular interest in recent
years.11–18 As deduced from autoradiographic studies, the high-
affinity Ang IV binding site but not the AT1 receptor is abundant
* Corresponding author. Tel.: +46 18 471 4141; fax: +46 18 501920.
E-mail address: Mathias.Hallberg@farmbio.uu.se (M. Hallberg).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.046

H. Andersson et al. / Bioorg. Med. Chem. 16 (2008) 6924–6935
6925
the native AP-N nor to the AP-N apo-enzyme.29 A receptor sub-
type that has been demonstrated to be involved at least partially
in the mediation of the observed cognitive effects of Ang IV is
the D2 dopamine receptor. It was clearly shown that D2 receptor
blockade abolished all cognitive effects mediated by Ang IV.30
Hence, it was proposed that Ang IV promotes release of dopa-
mine and possibly also acetylcholine in vivo31 and that the po-
tential liberation of these transmitter substances might play a
fundamental role downstream from IRAP/AT4 for the enhance-
ment of the cognitive effects observed.30 However, the link be-
tween Ang IV analogues and the IRAP/AT4 receptor in relation
to dopamine and the D2 receptor is still not clear, but the close
location of the IRAP/AT4 and D2 receptors in brain areas associ-
ated with learning and memory should be favourable for interac-
tions.32–34 Furthermore, a recent study showed that the effect of
Ang IV on dopamine release was not a result of inhibition of the
IRAP and AP-N enzyme activity, hence it was hypothesized that
the effect is a result of activation of IRAP and/or AP-N acting as
receptors.35
Regardless of the detailed underlying mechanism, the IRAP/AT4
receptor has recently evolved into a new target for pharmaceuticals
aimed for treatment of various cognitive disorders. We are con-
vinced that non-peptidic metabolically stable drug-like Ang IV ana-
logues that efficiently can cross the blood–brain barrier would be
attractive as research tools and enable extensive studies of the im-
pact of Ang IV on the in vivo physiology in complex animal models.
Our long-term goal is to design and synthesize such ligands. In addi-
tion, non-peptidic ligands could possibly serve as potential lead
structures for further optimization in drug discovery programmes.
We herein report that incorporation of a 4-hydroxydiphenyl-
methane scaffold as a substitute for Tyr2, in combination with a
phenylacetic acid or benzoic acid scaffold as substitute for the
His4-Pro5-Phe6 amino acid residues in Ang IV deliver more drug-
like IRAP/AT4 receptor ligands, although with lower potency than
Ang IV both with regard to receptor binding and IRAP inhibition
(1–11, Table 1). Further truncations and/or internal deletions ren-
der compounds that are essentially inactive (12–15, Table 1). A
comparison of the Ki values from the receptor binding and IRAP
inhibition assays indicates some differences in the structure–activ-
ity relationships (SAR).
Building blocks 21a42 and 21b37,38 (Scheme 2) were prepared
essentially according to a reported three-step procedure.37,38 Ini-
tially, a solvent-free protocol43 was used to obtain the brominated
intermediates ethyl 2-(bromomethyl)benzoate and ethyl 2-(bro-
momethyl)phenylacetate, but the reaction was found to be slug-
gish and gave complex reaction mixtures. Therefore, a protocol
employing methyl acetate as a solvent and microwave-assisted
heating was modified and applied.44 The commercially available
ethyl 2-methylbenzoate and ethyl 2-methylphenylacetate were re-
acted with N-bromosuccinimide (NBS) and 2,20-azobis(2-methyl-
propionitrile) (AIBN) in MeCN at 90 °C for 15 min to obtain ethyl
2-(bromomethyl)benzoate and ethyl 2-(bromomethyl)phenylace-
tate. The transformations of the bromomethyl groups into the cor-
responding azidomethyl groups were achieved by treatment with
NaN3 in DMF. Finally, the esters were hydrolyzed with LiOH in
THF/MeOH/H2O to obtain the corresponding carboxylic acids 21a
and 21b.
The target angiotensin IV analogues 2–15 were prepared by
manual solid-phase synthesis techniques using Fmoc protection
as described previously (Scheme 2).45 Initial coupling of 21a and
21b to 2-chlorotrityl chloride resin furnished 22a and 22b,46 which
were further elongated with
L-proline (23a and 23b),
L-leucine
(24a and 24b),
L
-isoleucine (25a and 25b) and 20 (26b). The elon-
gation was performed by a slightly modified literature procedure37
involving addition of tributylphosphine to a mixture of resin bound
azide and activated amino acid in CH2Cl2 or 20 in CH2Cl2/MeCN.
Prolonged reaction times were used for the subsequent couplings
of 20,
L-valine and L-norleucine as these reactions were expected
to proceed slowly. Analogues 1 and 15 were prepared in a similar
fashion by consecutive coupling of 20 and L-valine to 22c and 22d,
respectively.
The Fmoc-protected scaffold 20 was used in the pseudopeptides
to replace the Tyr2, Val1-Tyr2, Tyr2-Ile3 or Val1-Tyr2-Ile3 residues in
Ang IV. The building blocks 21a and 21b were utilized in com-
pounds 2–14 as
c
-turn mimicing scaffolds to replace His4-Pro5-
Phe6 in Ang IV.
2.2. Biochemical evaluation
The Ang IV analogues were tested and compared for their bind-
ing affinity to IRAP natively expressed in CHO-K1 cells as well as
their ability to inhibit the catalytic activity of recombinant human
IRAP and aminopeptidase N transiently transfected in HEK293
cells. The chemical structures, the binding affinities and protease
inhibition data of the compounds are presented in Table 1.
Our long-term objective is to develop drug-like Ang IV mimetics
that exhibit a high metabolic stability and oral bioavailability. We
foresee that such compounds, characterized by a considerably
longer duration of action than the parent peptide Ang IV, would
serve as attractive research tools, in particular when experiments
in complex animal models are performed. One approach to peptide
mimetics is to induce constrains by cyclizations and in those cases
where potent cyclized peptides are identified, proceed by conduct-
ing a conformational analysis to determine the bioactive confor-
mation of the target peptide. Subsequent displacement of the
peptide backbone with proper organic scaffolds, which mimic the
secondary structure of the bioactive conformation, followed by
structural optimization should eventually provide drug-like pep-
tide mimetics.47,48
2. Results and discussion
2.1. Synthesis of Ang IV analogues
The scaffolds utilized in the synthesis of the target analogues
(1–15, Table 1) were prepared via modifications of previously re-
ported procedures.36–38 The diphenylmethane scaffold 2036 was
synthesized according to the route outlined in Scheme 1. The first
step was conducted by a Negishi coupling39,40 using the acid chlo-
ride of the commercially available benzoic acid and the organozinc
reagent of p-bromoanisole. Conversion of p-bromoanisole to the
corresponding Grignard reagent followed by transmetallation
afforded the organozinc reagent which was used immediately. A
solid supported scavenger, 3-(1-thioureido)propyl-functionalized
silica gel,41 was used prior to the reduction of ketone 1636 to avoid
palladium-catalyzed side reactions. Triethylsilane and triflic acid in
TFA/CH2Cl2 were employed for the quantitative reduction provid-
ing 17.36 The hydrolysis of the ester was performed with LiOH in
THF/H2O/MeOH affording compound 1836 in a quantitative yield.
Compound 1936 was obtained by hydrogenation of the nitro group
using Pd/C in absolute ethanol followed by Fmoc protection of the
resulting aniline. Demethylation of the p-methoxy group to pro-
duce the corresponding phenol 2036 was accomplished with boron
trifluoride-methyl sulfide complex in CH2Cl2.
Recently, we demonstrated that 1 to 3 cyclization of the N-ter-
minal of Ang IV, for example, oxidative cyclization of Cys1 with
Cys3 where Val1 and Ile3 in Ang IV had been displaced, delivered
a peptide with an approximately 1000-fold lower binding affinity
than Ang IV itself.49 Since Cys1/Cys3 cyclized peptides preferen-
tially adopt
c-turns, it was concluded that it is not likely that a
c-turn is present in the N-terminal of Ang IV when the peptide is

6926
H. Andersson et al. / Bioorg. Med. Chem. 16 (2008) 6924–6935
Table 1
Structures, binding affinities and inhibition activities of compounds 1–15 and Ang IV
Compound
Structure
Binding affinity
Ki SDa
M)
IRAP enzyme inhibition
Ki SDb
M)
AP-N enzyme inhibition
Ki SDc
M)
(
l
(l
(l
Ang IVd
0.007 0.003
6.8 1.0
13 0.3
0.06 0.04
1.2 0.6
12 3.9
7.4 0.0
6.3 0.0
3.3 0.9
3.7 1.1
3.9 2.5
1.3 0.5
0.83 0.10
5.0 0.3
55 8.4
49 1.9
17 2.5
11 0.5
10 0.6
16 3.1
20 4.0
1
2
3
4
5
6
7
8
14 0.6
2.5 0.1
1.4 0.1
3.9 0.1
3.1 0.2
12 1.1

H. Andersson et al. / Bioorg. Med. Chem. 16 (2008) 6924–6935
6927
Table 1 (continued)
Compound
Structure
Binding affinity
Ki SDa
M)
IRAP enzyme inhibition
AP-N enzyme inhibition
(
l
Ki SDb
(l
M)
Ki SDc
(lM)
9
8.9 1.7
1.3 0.5
21 1.6
10
2.3 0.2
1.8 1.8
17 2.5
11
12
13
14
15
1.0 0.1
1.4 0.6
97 38
16 0.3
35 4.9
76 7.0
13 1.1
>100
>100
86 7.9
33 5.2
>100
>100
>100
69 7.7
SD, standard deviation.
a
[
125I]Angiotensin IV competition binding in CHO-K1 cell membranes.
b
c
Evaluated in an enzyme assay comprising recombinant human IRAP, transiently transfected in HEK293 cells.
Evaluated in an enzyme assay comprising recombinant human AP-N, transiently transfected in HEK293 cells.
Data for Ang IV are taken from Ref. 29.
d
binding to its target protein. However, by applying larger ring sys-
tems (i.e., Cyc1/Hcy3, Hcy1/Cys3 and Hcy1/Hcy3 cyclizations) there-
by providing higher conformational flexibility more potent ligands
could be obtained.49
Prior to the incorporation of more complex backbone mimetics
to replace residues 1–3, we wanted to assess if a simpler one-res-
idue backbone mimetic could be successfully employed. A series of
derivatives comprising a 4-hydroxydiphenylmethane scaffold to
replace the Tyr2 residue in Ang IV were designed and synthesized.
All except analogue 1 contain only two or less amino acid residues.
Diphenylmethane is one of the most common scaffolds in drugs.
The 4-hydroxydiphenylmethane fragment contains this privileged
structure50 and has previously been incorporated to replace
the amino acid residues centred at tyrosine in the octapeptide

6928
H. Andersson et al. / Bioorg. Med. Chem. 16 (2008) 6924–6935
terminal are in fact accepted49 but as apparent from the present
study small alterations of the backbone can exert a dramatic im-
pact on the bioactivity. Efforts, including modelling as were previ-
ously successfully applied for the C-terminal to determine the
active conformation will now be devoted to the identification of
optimal structural elements in the N-terminal of Ang IV.
Regarding modifications of the C-terminal part of Ang IV, we
previously reported that replacement of His4-Pro5-Phe6 in Ang IV
with a 2-aminomethylphenylacetic acid moiety delivered a ligand
with an equipotent binding affinity as compared to Ang IV when
the cortex membrane binding assay was employed.45 In analogy,
we have now found that 1 can be subjected to the same transfor-
mation with essentially no loss of activity, concerning neither
receptor affinity nor enzyme inhibition capacity, cf. 1 and 9. A
small loss of activity was encountered with a C-terminal benzoic
acid instead of the phenylacetic acid fragment, cf. 8 and 9. In gen-
eral, it was observed that that the benzoic acid derivatives were
less active than the corresponding phenylacetic acid derivatives.
The importance of the C-terminal part was demonstrated by the
truncated analogue 15, the binding affinity was lost and the en-
zyme inhibition dropped 60 times when the benzoic and phenyla-
cetic acid scaffolds were deleted, cf. 8, 9 and 15. The attempts to
impose more steric constrain by replacing Ile3 of 8 and 9 by proline
provided less active compounds (2 and 3) in particular with regard
to the enzyme inhibition. Both of the proline derivatives 2 and 3
are less active than 1 in particular with regard to AP-N inhibition.
Replacement of the isopropyl side chain of Val1 by a non-branched
butyl group, that is, norleucine, delivered more efficient ligands to
the IRAP/AT4 receptor, cf. 8 with 10 and 9 with 11, respectively.
The beneficial effect of a norleucine residue in position 1 in Ang
IV analogues is well established from recent binding studies51,52
and we assume therefore that the novel less peptidic ligands bind
to the receptor protein essentially in the same fashion as the native
hexapeptide does. In addition, with proline in position 3, a norleu-
cine residue in position 1 was favourable also for IRAP inhibition,
cf. 2 with 4 and 3 with 5, respectively. However, with isoleucine
in position 3, replacing valine by norleucine seemed to exert only
minor influences on the enzyme inhibition, cf. 8 with 10 and 9 with
11. Concerning position 3, a one carbon migration of a methyl
group, that is, a leucine instead of an isoleucine, had an unfavour-
Scheme 1. Reagents: (a) i—SOCl2; ii—4-MeOC6H4ZnCl, PdCl2(PPh3)2, THF; iii—3-(1-
thioureido)propyl-functionalized silica gel, THF; (b) Et3SiH, CF3SO2OH, TFA, DCM;
(c) LiOH, THF/MeOH/H2O; (d) i—H2, Pd/C, EtOH; ii—Fmoc-Cl, Na2CO3 (aq), dioxane;
(e) BF3ꢀS(CH3)2, DCM.
angiotensin II. Potent pseudopeptides with high affinity for the AT2
receptor were identified.36 However, we found that incorporation
of a 4-hydroxydiphenylmethane scaffold encompassing simply an
N-terminal aminomethyl group to substitute Val1-Tyr2 or Val1-
Tyr2-Ile3 in Ang IV rendered, in experiments where porcine frontal
cortex membranes were used, ligands that were 1000-fold less po-
tent than Ang IV or inactive, respectively.45 Similarly, with the ani-
lide-based 4-hydroxydiphenylmethane scaffold employed in the
present study we likewise obtained a 1000-fold drop in binding
affinity as compared to Ang IV, despite the presence of both Val1
and Ile3 (cf. Ang IV and 1). Notably though, a comparison of the
IRAP activity inhibition data revealed that 1 is only 20 times less
potent than Ang IV. Similarly, 1 is only six times less active as an
AP-N inhibitor compared to Ang IV. The incorporation of the 4-
hydroxydiphenylmethane scaffold in Ang IV to afford 1 rendered
a considerably less bioactive ligand suggesting that the aromatic
ring system applied herein induces constrain leading to unfavour-
able spatial arrangement of the side chains of the N-terminal. The
extended back bone and non-optimal locations of the peptide
bonds of 1 can provide alternative rationales for the lower activity.
As we recently demonstrated some large macrocylic rings in the N-
Scheme 2. Reagents: (a) i—NBS, AIBN, MeCN; (ii) NaN3, DMF; (iii) LiOH, THF/MeOH/H2O; (b) 2-chlorotrityl chloride resin, DIEA, CH2Cl2; (c) i—Fmoc-
CH2Cl2; ii—PBu3; (d) i—20, DIPCDI, HOBt, CH2Cl2/MeCN; ii—PBu3; (e) i—20% piperidine/DMF; ii—20, PyBOP, DIEA, DMF; (f) i—20% piperidine/DMF; ii—Fmoc-
L
-Xaa-OH, DIPCDI, HOBt,
-Xaa-OH, PyBOP,
L
DIEA, DMF; (g) i—20% piperidine/DMF; ii—Et3SiH, TFA/H2O.

H. Andersson et al. / Bioorg. Med. Chem. 16 (2008) 6924–6935
6929
able impact on both affinity and IRAP inhibition, cf. 6 with 10 and 7
with 11, respectively. Thus, it appears that both positions 1 and 3
in this series of ligands are important for the molecular recognition
and that even minor structural modifications of the lipophilic side
chains alter the bioactivity.
(0.2 mm, Merck) and the analytes were visualized by UV-light
and iodine. Column chromatography was performed using silica
gel 60 (40–63
corded on a GC–MS instrument equipped with a CP-Sil 8 CB capil-
lary column (30 m ꢁ 0.25 mm, 0.25 m) utilizing electron impact
lm, Merck). Low resolution mass spectra were re-
l
The weak potency of analogues 1–11 confirms the importance
of the N-terminal part both regarding binding and inhibi-
(EI) at an ionization energy of 70 eV. The oven temperature (GC)
was 40–300 °C. Analytical RP-HPLC-MS was performed on a Gilson
HPLC system with a Finnigan AQA quadrupol mass spectrometer
using an Onyx monolithic C18 column (50 ꢁ 4.6 mm) with gradi-
ents of MeCN/H2O (0.05% HCOOH) at a flow rate of 4 mL/min. Both
UV (DAD, 214 and 254 nm) and MS (ESI) detection were utilized.
Preparative RP-HPLC was performed using a Zorbax SB-C8 column
tion.49,51–54 These analogues incorporate
a large 4-dihydr-
oxidiphenylmethane scaffold in place of tyrosine at position 2. To
elucidate whether a tyrosine containing di- or tripeptide (i.e.,
Val1-Tyr2, Tyr2-Ile3 or Val1-Tyr2-Ile3) could be replaced by the
diphenylmethane scaffold whilst still retaining the activity, an
additional set of analogues (12–14) were synthesized. Although
the analogues containing four units (2–11) were found to be more
potent, the three and two unit analogues contributed some addi-
tional information. The importance of the amino acid residue in po-
sition 1 was reflected by the N-terminally truncated analogue 12,
which was inactive. Deletion of the residue in position 3 produced
the least negative outcome. Thus, analogue 13 showed a 10-fold
drop in enzyme inhibition and a 100-fold lower binding affinity
compared to the best compounds in the series of four unit ana-
logues (2–11). C-Terminal truncation of analogue 13 produced a
small non-peptidic compound (14) consisting of the 4-hydroxydi-
phenylmethane scaffold connected to the 2-(aminomethyl)phenyl-
acetic acid moiety. Whereas no binding affinity was encountered it
acted as a weak IRAP inhibitor.
(5
a flow rate of 5 mL/min and UV detection at 230 nm. The pseudo-
peptide purities were determined on an ACE 5 C18 column (5 m,
4.6 ꢁ 50 mm) and an ACE 5 Phenyl column (5 m, 4.6 ꢁ 50 mm)
lm, 21.2 ꢁ 150 mm) with gradients of MeCN/H2O (0.1% TFA) at
l
l
using the same buffer system at a flow rate of 2 mL/min and with
UV detection at 220 nm. NMR spectra were recorded on a Varian
Mercury plus spectrometer (1H at 399.8 MHz and 13C at
100.5 MHz or 1H at 399.9 MHz and 13C at 100.6 MHz) at ambient
temperature. Chemical shifts (d) are reported in ppm referenced
indirectly to TMS via the solvent residual signal. Exact molecular
masses were determined on a Micromass Q-Tof2 mass spectrome-
ter equipped with an electrospray ion source at the Department of
Pharmaceutical Biosciences, Uppsala University, Sweden. Amino
acid analyses were performed at the Department of Biochemistry
and Organic Chemistry, Uppsala University, Sweden. THF was
freshly distilled over Na/benzophenone, CH2Cl2 was distilled over
calcium hydride and N-bromosuccinimide (NBS) was recrystallized
from acetic acid. Other chemicals were used without further puri-
fication. Compounds 16–20,36 21a42 and 21b37,38 are known. Char-
acterization data were consistent with those previously reported.
Ang IV displays moderate selectivity for IRAP compared to AP-
N.29 More selective inhibitors and/or ligands could serve as valu-
able tools for investigation of the involvement of IRAP and AP-N
in mediating the effects of Ang IV. The present analogues exhibited
the similar selectivity as Ang IV.
3. Conclusions
4.1.1. Methyl 3-(4-methoxy-benzoyl)-5-nitrobenzoate36 (16)
5-Nitroisophthalic acid monomethyl ester (7.88 g, 35.0 mmol)
was refluxed in 70 mL thionyl chloride for 2 h. After evaporation
of the thionyl chloride, 100 mL dry CH2Cl2 was added and evapo-
rated. The resulting acid chloride was kept under vacuum over-
night. A dry three-necked round-bottomed flask was charged
with magnesium turnings (919 mg, 37.8 mmol), sealed with a sep-
tum, evacuated and filled with nitrogen. THF (28 mL), p-bromoani-
sole (4.38 mL, 35 mmol) and a catalytic amount of methyl iodide
(10 droplets) were added sequentially under stirring. The temper-
ature of the spontaneously warmed reaction mixture was slowly
decreasing. After 40 min it was heated to 60 °C and kept at that
temperature for approximately 3 h. The freshly prepared Grignard
reagent was allowed to attain ambient temperature and subse-
quently added over a 15 min period to a stirred solution of zinc
chloride (5.01 g, 36.8 mmol) in 28 mL dry THF at 0 °C. The reaction
mixture was stirred at 0 °C for 15 min and then at room tempera-
ture for approximately 4 h. The resulting slurry of the precipitated
white organozinc reagent was cooled to 0 °C and dichlorobis(tri-
phenylphosphine)palladium (737 mg, 1.05 mmol) was added. The
reaction vessel was flushed with nitrogen and a solution of the pre-
viously synthesized acid chloride in 40 mL dry THF was added
dropwise. The reaction mixture was stirred at room temperature
over night (16 h), poured into 300 mL 0.5 M HCl and extracted with
EtOAc (3ꢁ 200 mL). The combined organic phases were washed
with H2O (150 mL), dried over MgSO4, filtered and evaporated.
Purification by column chromatography (i-hexane/EtOAc 17:3–
13:4, and subsequently, toluene/EtOAc 96:4) gave 16 as a yellow
solid (4.99 g). To eliminate traces of palladium, 16 (3.58 g) was dis-
solved in 55 mL THF and heated to 60 °C for 80 min in the presence
of 3-(1-thioureido)propyl-functionalized silica gel (3.82 mg, load-
ing: 1.1 mmol/g, 200–400 mesh). The functionalized silica was
subsequently filtered off and washed with 50 mL THF. The
Ang IV peptide mimetics that retain binding affinity to the IRAP/
AT4 receptor and with a capacity to inhibit the proteolytic activity
of IRAP have been designed and synthesized. The most potent li-
gand in the series, compound 11, was found to be 140 times less
active than Ang IV in the binding assay but only about 25 times less
efficient as an IRAP inhibitor. Concerning structure–activity rela-
tionships, some differences were observed for receptor binding
and enzyme inhibition. However, it is difficult to explain that the
compounds in general exhibit an equal or higher potency in the
IRAP inhibition assay than in the binding assay whereas Ang IV
has the reverse profile. Characteristic features of the new pseudo-
peptides are the benzene ring replacing the methine carbon of Tyr2
and a phenylacetic acid or a benzoic acid replacing His4-Pro5-Phe6.
Further truncations and/or deletions were deleterious for activity.
The best Ang IV analogues in this study comprise only two amino
acid residues and are consequently much less peptidic in character
than the majority of the Ang IV pseudopeptides previously re-
ported in the literature. Ang IV peptide mimetics are foreseen to
be of considerable interest as research tools and the ligands pre-
sented herein will be subjected to further optimization with the
primary objectives to improve potency as well as stability to pro-
tease degradation.
4. Experimental
4.1. Chemistry
Microwave-heated reactions were performed in a Smith Syn-
thesizerTM (Biotage AB, Uppsala, Sweden) producing controlled irra-
diation at 2450 MHz. Thin-layer chromatography (TLC) was
conducted on aluminium sheets precoated with silica gel 60 F254

6930
H. Andersson et al. / Bioorg. Med. Chem. 16 (2008) 6924–6935
combined filtrates were evaporated to give 1636 as a pale yellow
solid (3.55 g, 45% overall).
was heated to 90 °C by microwave irradiation for 15 min, cooled
to room temperature, transferred to a round-bottomed flask and
concentrated under reduced pressure. Subsequent precipitation
of succinimide by i-hexane/CH2Cl2, filtration, evaporation and puri-
fication by column chromatography (i-hexane/ether 98:2) gave
ethyl 2-(bromomethyl)benzoate as a pale yellow oil (1.97 g, 67%).
1H NMR (CDCl3) d 7.96 (dd, 7.8, 1.4 Hz, 1H), 7.50–7.43 (m, 2H),
7.36 (ddd, J = 7.8, 6.8, 2.0 Hz, 1H), 4.95 (s, 2H), 4.40 (q, J = 7.1 Hz,
2H), 1.42 (t, J = 7.1 Hz, 3H). 13C NMR (CDCl3) d 166.7, 139.1,
132.5, 131.7, 131.3, 129.6, 128.6, 61.4, 31.7, 14.3. EI+-MS m/z 243
(M+). The residual compound obtained above (1.94 g, 7.99 mmol)
was reacted with NaN3 (0.62 g, 9.58 mmol) in 10 mL DMF at room
temperature for 22 h. The reaction was quenched with H2O and ex-
tracted with EtOAc. The combined organic layers were washed with
brine and H2O, dried over Na2SO4, filtered and evaporated. Purifica-
tion by column chromatography (i-hexane/EtOAc 95:5) gave ethyl
2-(azidomethyl)benzoate as a colourless oil (1.56 g, 95%). 1H NMR
(CDCl3) d 8.02 (dd, J = 7.8, 1.5 Hz, 1H), 7.55 (ddd, J = 7.5, 7.5, 1.5 Hz,
1H), 7.49 (ddd, J = 7.8, 1.5, 0.5 Hz, 1H), 7.40 (ddd, J = 7.5, 7.5,
1.5 Hz, 1H), 4.82 (s, 2H), 4.39 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz,
3H). 13C NMR (CDCl3) d 166.9, 137.3, 132.7, 131.2, 129.9, 129.3,
128.2, 61.4, 53.2, 14.4. LiOH (0.89 g, 37.0 mmol) was added to a solu-
tion of the residual compound obtained above (1.52 g, 7.41 mmol) in
THF(14 mL), MeOH(2 mL)andH2O(4 mL).Afterstirringfor17 h, the
reaction mixture was diluted with CH2Cl2 and H2O, and extracted
with CH2Cl2. The aqueous phase was acidified with 3 M HCl and ex-
tracted with CH2Cl2. The combined organic phases were washed
with brine and H2O, dried over Na2SO4, filtered and evaporated to
give 21a42 as a white solid (1.24 g, 95%). 1H NMR (acetone-d6) d
8.08 (dd, J = 7.8, 1.5 Hz, 1H), 7.64 (ddd, J = 7.5, 7.5, 1.5 Hz, 2H), 7.56
(dd, J = 7.8, 1.5 Hz, 1H), 7.50 (ddd, J = 7.5, 7.5, 1.5 Hz, 2H), 4.87 (s,
2H). 13C NMR (acetone-d6) d 168.2, 138.3, 133.5, 132.1, 131.2,
130.4, 129.1, 53.4. ESIꢂ-MS m/z 176.0 (MꢂH)ꢂ, 222.0
(M+HCOOHꢂH)ꢂ, 353.1 (2MꢂH)ꢂ, 572.3 (3MꢂH)ꢂ.
4.1.2. Methyl 3-(4-methoxybenzyl)-5-nitrobenzoate36 (17)
Trifluoroacetic acid (TFA) (8.45 mL, 110 mmol) and triflic acid
(0.10 mL, 1.1 mmol) were added to a stirred solution of 16 (3.48 g,
11 mmol) in 25 mL CH2Cl2 at 0 °C. After dropwise addition of trieth-
ylsilane (5.59 mL, 35 mmol), the reaction mixture was allowed to at-
tain ambient temperature. It was stirred for 3 h and then slowly
poured into a cold saturated solution of NaHCO3 (100 mL). The aque-
ous phase was extracted with CH2Cl2 (2ꢁ 50 mL) and the combined
organic phases were washed with H2O (2ꢁ 75 mL), dried over
MgSO4, filtered and evaporated. Excessive triethylsilane was re-
moved by repeated addition and evaporation of toluene. Compound
1736 was collected as a pale yellow solid (3.29 g, 99%).
4.1.3. 3-(4-Methoxybenzyl)-5-nitrobenzoic acid36 (18)
LiOH (1.30 g, 54.2 mmol) was added to a solution of 17 (3.25 g,
10.8 mmol) in THF (130 mL), MeOH (43 mL) and H2O (43 mL). After
stirring over night the reaction mixture was concentrated, diluted
with H2O and acidified with 1 M HCl. EtOAc (250 mL) was added
and the two phases were separated. The aqueous phase was ex-
tracted with EtOAc (50 mL) and the combined organic phases were
washed with brine (2ꢁ 100 mL) and H2O (100 mL), dried over
MgSO4, filteredand evaporatedto give1836 as a solid(3.09 g, quant.).
4.1.4. 3-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-(4-
methoxybenzyl)benzoic acid36 (19)
A mixture of compound 18 (3.07 g, 10.7 mmol) and 10% Pd/C
(1.14 g, 1.07 mmol) in 150 mL absolute ethanol was stirred under
hydrogen at atmospheric pressure and room temperature for
approximately 5 h. The crude product obtained after filtration
and evaporation, when analyzed by LC–MS, was considered to be
of sufficient purity to be used in the next step without further puri-
fication. An aqueous solution of Na2CO3 (0.3 M, 125 mL) was
slowly added to a solution of the aniline in 100 mL dioxane at
0 °C. 9-Fluorenylmethyl chloroformate (Fmoc-Cl) (3.36 g,
11.8 mmol) in 100 mL dioxane was added dropwise, whereafter
the reaction mixture was allowed to reach ambient temperature
and left under continuous stirring for approximately 44 h. The
reaction mixture was slowly acidified with 1 M HCl and EtOAc
(200 mL) was added. The aqueous phase was extracted with EtOAc
(2ꢁ 100 mL) and the combined organic phases were washed with
brine (2ꢁ 100 mL) and H2O (2ꢁ 100 mL), dried over MgSO4,
filtered and evaporated. Purification by column chromatography
(i-hexane/EtOAc/HCOOH 74:25:1) and an additional extraction
using EtOAc and H2O gave 1936 as a solid (4.52 g, 88%).
4.1.7. 2-(Azidomethyl)phenylacetic acid37,38 (21b)
2-(Azidomethyl)phenylacetic acid was prepared according to the
procedure described above. Ethyl 2-methylphenylacetate (2.14 mL,
12.0 mmol), NBS (2.35 g, 13.2 mmol), AIBN (98.5 mg, 0.60 mmol)
and MeCN (18 mL) were used in the first step. Ethyl 2-(bromo-
methyl)phenylacetate was obtained as a pale yellow oil (2.39 g,
77%). 1H NMR (CDCl3) d 7.37 (m, 1H), 7.30–7.24 (m, 3H), 4.60 (s,
2H), 4.16 (q, J = 7.2 Hz, 2H), 3.79 (s, 2H), 1.26 (t, J = 7.2 Hz, 3H). 13
C
NMR (CDCl3) d 171.2, 136.5, 133.6, 131.4, 130.8, 129.3, 128.1, 61.3,
38.5, 31.9, 14.3. EI+-MS m/z 257 (M+). The residual compound ob-
tained above (2.19 g, 8.52 mmol), NaN3 (0.66 g, 10.2 mmol) and
DMF (10 mL) were used in the second step. Ethyl 2-(azido-
methyl)phenylacetate was obtained as a pale yellow oil (1.85 g).
1H NMR (CDCl3) d 7.34–7.29 (m, 4H), 4.42 (s, 2H), 4.15 (q,
J = 7.2 Hz, 2H), 3.71 (s, 2H), 1.25 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3)
d 171.2, 134.1, 133.3, 131.3, 130.0, 129.0, 127.9, 61.2, 52.9, 38.7,
14.3. The ethyl 2-(azidomethyl)phenylacetate obtained above
(1.85 g), LiOH (1.01 g, 42.2 mmol), THF (14 mL), MeOH (2 mL) and
H2O(4 mL)wereusedinthelaststep. Thecrudeproductwaspurified
by filtration trough a pad of silica followed by recrystallization (tol-
uene/ether 1:3) and column chromatography (i-hexane/EtOAc/
HCOOH 60:40:1) to afford 21b37,38 as a white solid (1.20 g, 73% over
two steps). 1H NMR (acetone-d6) d 7.43–7.29 (m, 4H), 4.54 (s, 2H),
3.77 (s, 2H). 13C NMR (acetone-d6) d 172.4, 135.4, 135.0, 132.1,
130.5, 129.4, 128.2, 53.1, 38.4. ESIꢂ-MS m/z 190.0 (MꢂH)ꢂ, 236.1
(M+HCOOHꢂH)ꢂ, 381.2 (2MꢂH)ꢂ, 572.3 (3MꢂH)ꢂ.
4.1.5. 3-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-(4-
hydroxybenzyl)benzoic acid36 (20)
Boron trifluoride-methyl sulfide complex (10.2 mL, 97 mmol)
was added dropwise to 19 (4.65 g, 9.70 mmol) in 145 mL dry CH2Cl2
at 0 °C under nitrogen. The reaction mixture was allowed to reach
ambient temperature and left under continuous stirring for 26 h.
The reaction was quenched by very slow addition of the mixture to
cold 0.5 M HCl under stirring. Subsequent extraction with EtOAc
was followed by washing with NaHCO3, brine and H2O, drying over
MgSO4, filtration and evaporation. Purification by column chroma-
tography (i-hexane/EtOAc/HCOOH 65:34:1 followed by EtOAc to
elute the product) gave 2036 as a solid (4.18 g, 93%).
4.1.6. 2-(Azidomethyl)benzoic acid42 (21a)
Ethyl 2-methylbenzoate (1.91 mL, 12.0 mmol), NBS (2.35 g,
13.2 mmol), AIBN (98.5 mg, 0.60 mmol) and MeCN (18 mL) were
added to a 20 mL Smith Process VialTM containing a stirring bar.
After capping and evacuation of the vial, the reaction mixture
4.1.8. 2-(Azidomethyl)benzoyl-2-chlorotrityl resin (22a)
2-(Azidomethyl)benzoic acid 21a (101 mg, 0.57 mmol) was re-
acted with 2-chlorotrityl chloride resin (584 mg, 0.86 mmol) in
CH2Cl2 (6 mL) in the presence of N,N-diisopropylethylamine (DIEA)

H. Andersson et al. / Bioorg. Med. Chem. 16 (2008) 6924–6935
6931
(0.39 mL, 2.24 mmol). After 4 h, MeOH (0.5 mL) was added and the
mixture was stirred for another 25 min. The resin was washed with
several portions of, in turn, CH2Cl2, DMF and CH2Cl2, and dried in va-
cuo overnight. Two batches were made and the resulting substitu-
tion degree was determined to 0.86 and 0.89 mmol/g based on the
weight increase.
771
(283 mg, 242
Reaction time: 6 h. Yield: 354 mg.
l
mol), DIPCDI (121
l
L, 771
l
mol), CH2Cl2 (15 mL), 22b
l
mol) and tributylphosphine (149 lL, 514 lmol).
4.2.3. Fmoc-Leu-2-(aminomethyl)benzoyl-2-chlorotrityl resin
(24a)
Resin 24a was prepared according to the general procedure
using Fmoc-Leu-OH (379 mg, 1.07 mmol), HOBt (145 mg,
4.1.9. 2-(Azidomethyl)phenylacetyl-2-chlorotrityl resin (22b)
2-(Azidomethyl)phenylacetic acid 21b (101 mg, 0.53 mmol)
was reacted with 2-chlorotrityl chloride resin (556 mg, 0.82 mmol)
in CH2Cl2 (6 mL) in the presence of DIEA (0.37 mL, 2.12 mmol) fol-
lowing the procedure described above. Two batches were made
and the resulting substitution degree was determined to 0.84
and 0.85 mmol/g from the weight increase. Another batch was
made using 2-(azidomethyl)phenylacetic acid 21b (104 mg,
0.54 mmol), 2-chlorotrityl chloride resin (534 mg, 0.79 mmol)
and DIEA (0.37 mL, 2.1 mmol). Substitution degree: 0.88 mmol/g.
1.07
(315 mg, 281
Reaction time: 4 h. Yield: 379 mg.
l
mol), DIPCDI (168
l
L, 1.07 mmol), CH2Cl2 (20 mL), 22a
lmol) and tributylphosphine (206
l
L, 710 mol).
l
4.2.4. Fmoc-Leu-2-(aminomethyl)phenylacetyl-2-chlorotrityl
resin (24b)
Resin 24b was prepared according to the general procedure
using Fmoc-Leu-OH (269 mg, 760
774 mol), DIPCDI (119 L, 761 mol), CH2Cl2 (20 mL), 22b
(305 mg, 258 mol) and tributylphosphine (147 L, 510 mol).
Reaction time: 4 h. Yield: 373 mg.
lmol), HOBt (105 mg,
l
l
l
l
l
l
4.1.10. Ile-His(Trt)-Pro-Phe-Wang resin (22c)
The Ile-His(Trt)-Pro-Phe-Wang resin was synthesized with a
Symphony instrument using Fmoc protection. The starting poly-
mer was Fmoc-Phe-Wang resin (297 mg, 0.25 mmol). Removal of
the Fmoc group was achieved by reaction with 20% piperidine in
4.2.5. Fmoc-Ile-2-(aminomethyl)benzoyl-2-chlorotrityl resin
(25a)
Resin 25a was prepared according to the general procedure
DMF (2ꢁ 2.5 mL, 5 + 10 min). The amino acids (125
coupled in DMF using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetram-
ethyluronium hexafluorophosphate (HBTU) (125 mol) in the
presence of N-methylmorpholine (NMM) (500 mol). Double cou-
l
mol) were
using Fmoc-Ile-OH (275 mg, 779
DIPCDI (122 L, 779 mol), CH2Cl2 (15 mL), 22a (301 mg,
260 mol) and tributylphosphine (150 L, 520 mol). Reaction
time: 6 h. Yield: 378 mg.
lmol), HOBt (105 mg, 779 lmol),
l
l
l
l
l
l
l
plings (2 ꢁ 30 min) were used for all amino acids. At the end of
each coupling cycle, the remaining amino groups were capped by
addition of 20% acetic anhydride in DMF (1.25 mL) to the reaction
mixture and allowing the reaction to proceed for 5 min. After com-
pletion of the synthesis the Fmoc group was removed and the par-
tially protected peptide resin was washed with several portions of
DMF and CH2Cl2 and then dried in a stream of nitrogen and in va-
cuo. The resulting substitution degree was determined to
0.55 mmol/g based on the weight increase.
4.2.6. Fmoc-Ile-2-(aminomethyl)phenylacetyl-2-chlorotrityl
resin (25b)
Resin 25b was prepared according to the general procedure
using Fmoc-Ile-OH (273 mg, 771
DIPCDI (121 L, 771 mol), CH2Cl2 (15 mL), 22b (301 mg,
257 mol) and tributylphosphine (149 L, 514 mol). Reaction
time: 6 h. Yield 381 mg.
lmol), HOBt (104 mg, 771 lmol),
l
l
l
l
l
4.2.7. 3-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-(4-
hydroxybenzyl)benzoyl-2-(aminomethyl)phenylacetyl-2-
chlorotrityl resin (26b)
4.1.11. Fmoc-Ile-2-chlorotrityl resin (22d)
Fmoc-Ile-OH (142 mg, 0.40 mmol) was reacted with 2-chloro-
trityl chloride resin (341 mg, 0.50 mmol) in CH2Cl2 (6 mL) in the
presence of DIEA (0.28 mL, 1.61 mmol) following the procedure de-
scribed for 22a. Substitution degree: 0.93 mmol/g.
Resin 26b was prepared according to the general procedure
using 20 (246 mg, 528
(82.2 L, 525 mol), CH2Cl2 (30 mL), MeCN (5 mL), 22b (399 mg,
351 mol) and tributylphosphine (203 L, 700 mol). Reaction
time: 5 h. Yield: 517 mg.
lmol), HOBt (77.7 mg, 575 lmol), DIPCDI
l
l
l
l
l
4.2. General procedure for the preparation of resins 23a, 23b,
24a, 24b, 25a, 25b and 26b
4.3. General synthesis of angiotensin IV analogues 1–15
A round-bottomed flask was charged with the appropriate ami-
no acid or 20, 1-hydroxybenzotriazole (HOBt) and N,N0-diisopro-
pylcarbodiimide (DIPCDI) in dry CH2Cl2 or CH2Cl2/MeCN. After
preactivation under nitrogen for 10 min, 22a or 22b was added,
followed by tributylphosphine after another 10 min. The reaction
mixture was stirred under nitrogen for 4–6 h. The resin was fil-
tered off and washed with several portions of, in turn, CH2Cl2,
DMF, MeOH and CH2Cl2, and finally dried in in vacuo overnight.
Resin 22c, 22d, 23a, 23b, 24a, 24b, 25a, 25b or 26b was weighed
into a 2 mL disposable syringe fitted with porous polyethylene fil-
ter. The Fmoc group was removed by treatment with 20% piperi-
dine in DMF (2ꢁ 1.5 mL, 10 + 15 min) and the polymer was
washed with DMF (5ꢁ 1.5 mL, 5 ꢁ 1 min). Coupling of 20 and/or
the appropriate amino acid was performed by agitation over night
in DMF (1.5 mL) using benzotriazole-1-yl-oxy-tris-pyrrolidino-
phosphonium hexafluorophosphate (PyBOP) in the presence of
DIEA. The resin was washed with DMF (5ꢁ 1.5 mL, 5 ꢁ 1 min)
and subsequently deprotected and washed as described above.
After completion of the coupling cycle, the resin was also washed
with several portions of, in turn, CH2Cl2, MeOH and CH2Cl2 before
it was dried in in vacuo. The pseudopeptide was cleaved by treat-
ment with TFA/H2O/triethylsilane (90:5:5 ca. 1 mL/100 mg resin)
for 2 h. The resin was filtered off and washed with TFA (4ꢁ 0.3–
0.5 mL). The filtrate was collected in a centrifuge tube and concen-
trated in a stream of nitrogen. Cold diethyl ether (ca. 13 mL) was
used to precipitate the product, which was collected by centrifuga-
tion, washed with cold diethyl ether (63ꢁ 7 mL) and dried. The
4.2.1. Fmoc-Pro-2-(aminomethyl)benzoyl-2-chlorotritylresin(23a)
Resin 23a was prepared according to the general procedure
using Fmoc-Pro-OH (263 mg, 779
779 mol), DIPCDI (122 L, 779 mol), CH2Cl2 (15 mL), 22a
(303 mg, 262 mol) and tributylphosphine (150 L, 520 mol).
Reaction time: 6 h. Yield: 373 mg.
lmol), HOBt (105 mg,
l
l
l
l
l
l
4.2.2. Fmoc-Pro-2-(aminomethyl)phenylacetyl-2-chlorotrityl
resin (23b)
Resin 23b was prepared according to the general procedure
using Fmoc-Pro-OH (260 mg, 771 lmol), HOBt (104 mg,

6932
H. Andersson et al. / Bioorg. Med. Chem. 16 (2008) 6924–6935
crude pseudopeptide was dissolved in MeCN/0.1% aqueous TFA, fil-
tered through a 0.45 m nylon membrane and purified in 1–2 runs
by RP-HPLC. Selected fractions were analyzed by RP-HPLC and RP-
HPLC-MS, and those containing pure product were pooled and
lyophilized.
Reaction time: 17 h and 18 h, respectively. Purification by prepara-
tive RP-HPLC gave 4 as a white solid (13.6 mg, 19%). 1H NMR
(CD3OD) d (3:1 mixture of rotamers, major rotamer reported)
7.96 (dd, J = 7.8, 1.4 Hz, 1H), 7.73 (m, 1H), 7.57 (m, 1H), 7.51 (m,
1H), 7.47 (m, 1H), 7.34 (m, 1H), 7.24 (m, 1H), 7.02 (m, 2H), 6.70
(m, 2H), 4.77 (app. s, 2H), 4.60 (dd, J = 8.1, 5.8 Hz, 1H), 3.96–3.91
(m, 3H), 3.62 (m, 1H), 3.51 (m, 1H), 2.32 (m, 1H), 2.05–1.81 (m,
5H), 1.44–1.35 (m, 4H), 0.95 (t, J = 7.2 Hz, 3H). 13C NMR (CD3OD)
d (major rotamer reported) 174.5, 171.7, 170.9, 168.7, 157.0,
145.2, 140.9, 139.2, 138.2, 133.3, 132.5, 132.0, 131.0, 129.8,
128.1, 124.8, 123.1, 117.7, 116.4, 62.2, 55.2, 51.7, 43.1, 41.7, 32.5,
31.1, 28.0, 26.3, 23.4, 14.1. One carbon signal in the aromatic area
is missing. HPLC purity: C18 column 99.5%, Phenyl column 99.7%.
HRMS (M+H+): 587.2873, C33H39N4O6 requires 587.2870. Amino
acid analysis: Nle, 1.01; Pro, 0.99 (77% peptide).
l
4.3.1. Analogue 1
Except for the initial Fmoc deprotection, the analogue was pre-
pared according to the general procedure using 22c (251 mg,
138
and DIEA (59.9
Fmoc-Val-OH (142 mg, 418
and DIEA (144 L, 829 mol) in the second. Reactiontime: 18 h
l
mol), 20 (80.2 mg, 172
L, 344 mol) in the first coupling followed by
mol), PyBOP (217 mg, 417 mol)
lmol), PyBOP (89.6 mg, 172 lmol)
l
l
l
l
l
l
for both couplings. Purification by preparative RP-HPLC gave 1 as
a white solid (11.3 mg, 25%). HPLC purity: C18 column 99.0%, Phe-
nyl column 99.1%. HRMS (M+H+): 837.4277, C45H57N8O8 requires
837.4299. Amino acid analysis: Val 1.05; Ile 0.99; His 0.98; Pro
0.98; Phe 1.00.
4.3.5. Analogue 5
The analogue was prepared according to the general procedure
using 23b (148 mg, 101
(92.5 mg, 178 mol) and DIEA (63.5
pling followed by Fmoc-Nle-OH (91.4 mg, 253
(132 mg, 253 mol) and DIEA (88.2 L, 506 mol) in the second.
l
mol), 20 (84.8 mg, 182
L, 364 mol) in the first cou-
mol), PyBOP
lmol), PyBOP
4.3.2. Analogue 2
l
l
l
The analogue was prepared according to the general procedure
l
using 23a (170 mg, 120
(95.8 mg, 184 mol) and DIEA (64.1
pling followed by Fmoc-Val-OH (100 mg, 294
(153 mg, 294 mol) and DIEA (103 L, 589 mol) in the second.
l
mol), 20 (85.7 mg, 184
L, 368 mol) in the first cou-
mol), PyBOP
l
mol), PyBOP
l
l
l
l
l
l
Reaction time: 16 h for both couplings. Purification by preparative
RP-HPLC gave 5 as a white solid (17.4 mg, 29%). 1H NMR (CD3OD) d
(3:1 mixture of rotamers, major rotamer reported) 7.73 (dd, J = 2.1,
1.6 Hz, 1H), 7.46 (dd, J = 2.1, 1.6 Hz, 1H), 7.40 (m, 1H), 7.28–7.21
(m, 4H), 7.02 (m, 2H), 6.70 (m, 2H), 4.54 (dd, J = 8.1, 5.8 Hz, 1H),
4.50 (d, J = 15.0 Hz, 1H), 4.46 (d, J = 15.0 Hz, 1H), 3.94–3.91 (m,
3H), 3.77 (d, J = 16.0 Hz, 1H), 3.73 (d, J = 16.0 Hz, 1H), 3.62 (m,
1H), 3.51 (m, 1H), 2.31 (m, 1H), 2.03–1.81 (m, 5H), 1.44–1.35 (m,
4H), 0.95 (t, J = 7.2 Hz, 3H). 13C NMR (CD3OD) d (major rotamer re-
ported) 175.4, 174.4, 171.6, 168.7, 157.0, 145.2, 139.2, 138.2, 138.1,
134.4, 132.5, 131.8, 131.0, 129.8, 128.6, 124.9, 123.1, 117.6, 116.4,
62.2, 55.2, 51.8, 42.0, 41.7, 39.1, 32.5, 31.2, 28.0, 26.4, 23.4, 14.1.
One carbon signal in the aromatic area is missing due to overlap-
ping signals. HPLC purity: C18 column 98.5%, Phenyl column
99.5%. HRMS (M+H+): 601.3038, C34H41N4O6 requires 601.3026.
Amino acid analysis: Nle, 1.01; Pro, 0.99 (59% peptide).
l
l
l
l
Reaction time: 17 h and 18 h, respectively. Purification by prepara-
tive RP-HPLC gave 2 as a white solid (5.1 mg, 7%). 1H NMR (CD3OD)
d (3:1 mixture of rotamers, major rotamer reported) 7.98 (dd,
J = 7.8, 1.4 Hz, 1H), 7.73 (m, 1H), 7.58 (m, 1H), 7.52 (m, 1H), 7.46
(m, 1H), 7.35 (m, 1H), 7.25 (m, 1H), 7.03 (m, 2H), 6.70 (m, 2H),
4.78 (app. s, 2H), 4.60 (dd, J = 8.2, 5.8 Hz, 1H), 3.92 (app. s, 2H),
3.73 (d, J = 5.9 Hz, 1H), 3.68 (m, 1H), 3.56 (m, 1H), 2.35–2.17 (m,
2H), 2.06–1.97 (m, 2H), 1.87 (m, 1H), 1.11 (d, J = 6.9 Hz, 3H), 1.08
(d, J = 6.9 Hz, 3H). HPLC purity: C18 column >99.9%, Phenyl column
98.3%. HRMS (M+H+): 573.2711, C32H37N4O6 requires 573.2713.
Amino acid analysis: Val, 1.13; Pro, 0.87 (72% peptide).
4.3.3. Analogue 3
The analogue was prepared according to the general procedure
using 23b (148 mg, 101
(92.5 mg, 178 mol) and DIEA (63.5
pling followed by Fmoc-Val-OH (99.4 mg, 293
(153 mg, 293 mol) and DIEA (102 L, 586 mol) in the second.
l
mol), 20 (84.8 mg, 182
L, 364 mol) in the first cou-
mol), PyBOP
l
mol), PyBOP
4.3.6. Analogue 6
l
l
l
The analogue was prepared according to the general procedure
l
using 24a (100 mg, 74.2
(130 mg, 249 mol) and DIEA (86.8
pling followed by Fmoc-Nle-OH (96.5 mg, 272
(130 mg, 249 mol) and DIEA (86.8 L, 498 mol) in the second.
l
mol), 20 (115 mg, 247
L, 498 mol) in the first cou-
mol), PyBOP
lmol), PyBOP
l
l
l
l
l
l
Reaction time: 16 h for both couplings. Purification by preparative
RP-HPLC gave 3 as a white solid (5.9 mg, 10%). 1H NMR (CD3OD) d
(3:1 mixture of rotamers, major rotamer reported) 7.73 (m, 1H),
7.46 (m, 1H), 7.39 (m, 1H), 7.29–7.20 (m, 4H), 7.02 (m, 2H), 6.70
(m, 2H), 4.54 (dd, J = 8.1, 5.8 Hz, 1H), 4.50 (d, J = 15.0 Hz, 1H),
4.46 (d, J = 15.0 Hz, 1H), 3.91 (app. s, 2H), 3.75 (app. s, 2H), 3.73
(d, J = 5.9 Hz, 1H), 3.63 (m, 1H), 3.52 (m, 1H), 2.35–2.17 (m, 2H),
2.05–1.95 (m, 2H), 1.86 (m, 1H), 1.11 (d, J = 6.9 Hz, 3H), 1.08 (d,
J = 6.9 Hz, 3H). 13C NMR (CD3OD) d (major rotamer reported)
175.4, 174.4, 171.6, 168.0, 157.0, 145.2, 139.1, 138.2, 138.0,
134.4, 132.5, 131.8, 131.0, 129.8, 128.6, 124.9, 123.1, 117.7,
116.4, 62.2, 60.5, 51.8, 42.1, 41.7, 39.1, 31.7, 31.2, 26.4, 19.0,
17.8. One carbon signal in the aromatic area is missing due to over-
lapping signals. HPLC purity: C18 column 99.4%, Phenyl column
99.4%. HRMS (M+H+): 587.2854, C33H39N4O6 requires 587.2870.
Amino acid analysis: Val, 1.04; Pro, 0.96 (77% peptide).
l
l
l
l
Reaction time: 15 h and 17 h, respectively. Purification by prepara-
tive RP-HPLC gave 6 as a white solid (6.6 mg, 15%). 1H NMR
(CD3OD) d (9:1 mixture of rotamers, major rotamer reported)
7.98 (m, 1H), 7.91 (dd, J = 2.1, 1.6 Hz, 1H), 7.52–7.48 (m, 4H),
7.35 (m, 1H), 7.04 (m, 2H), 6.71 (m, 2H), 4.73 (m, 2H), 4.64 (m,
1H), 3.95–3.91 (m, 3H), 1.99–1.84 (m, 2H), 1.78–1.67 (m, 3H),
1.47–1.36 (m, 4H), 1.00–0.93 (m, 9H). 13C NMR (CD3OD) d (major
rotamer reported) 174.8, 170.6, 170.2, 168.8, 157.0, 145.2, 141.0,
139.3, 136.5, 133.5, 132.5, 132.2, 131.0, 130.1, 128.3, 125.1,
124.6, 118.2, 116.4, 55.2, 54.1, 43.2, 41.7, 32.5, 28.0, 26.2, 23.4,
22.0, 14.1. One carbon signal in the aromatic area is missing and
two carbon signals in the aliphatic area are missing due to overlap-
ping signals. HPLC purity: C18 column >99.9%, Phenyl column
99.5%. HRMS (M+H+): 603.3172, C34H43N4O6 requires 603.3183.
Amino acid analysis: Nle, 1.01; Leu, 0.99 (89% peptide).
4.3.4. Analogue 4
The analogue was prepared according to the general procedure
4.3.7. Analogue 7
using 23a (170 mg, 120
(95.8 mg, 184 mol) and DIEA (64.1
pling followed by Fmoc-Nle-OH (104 mg, 294
(153 mg, 294 mol) and DIEA (103 L, 589 mol) in the second.
l
mol), 20 (85.7 mg, 184
L, 368 mol) in the first cou-
mol), PyBOP
l
mol), PyBOP
The analogue was prepared according to the general procedure
l
l
l
using 24b (151 mg, 101
(96.7 mg, 187 mol) and DIEA (130
pling followed by Fmoc-Nle-OH (133 mg, 374 l
l
mol), 20 (174 mg, 374
L, 74.8 mol) in the first cou-
mol), PyBOP
lmol), PyBOP
l
l
l
l
l
l
l

H. Andersson et al. / Bioorg. Med. Chem. 16 (2008) 6924–6935
6933
(196 mg, 374
l
mol) and DIEA (130
l
L, 748
l
mol) in the second.
7.99 (m, 1H), 7.90 (dd, J = 2.1, 1.6 Hz, 1H), 7.52 (dd, J = 2.1, 1.5 Hz,
1H), 7.49–7.45 (m, 3H), 7.35 (m, 1H), 7.03 (m, 2H), 6.71 (m, 2H),
4.78 (d, J = 15.5 Hz, 1H), 4.73 (d, J = 15.5 Hz, 1H), 4.42 (d,
J = 8.5 Hz, 2H), 3.95–3.92 (m, 3H), 2.00–1.84 (m, 3H), 1.59 (ddq,
J = 13.6, 3.5, 7.5 Hz, 1H), 1.47–1.36 (m, 4H), 1.24 (ddq, J = 13.6,
9.0, 7.4 Hz, 1H), 0.97–0.90 (m, 9H). 13C NMR (CD3OD) d (major rot-
amer reported) 173.8, 170.4, 170.2, 168.8, 157.0, 145.3, 141.0,
139.3, 136.6, 133.5, 132.4, 132.2, 131.0, 130.3, 128.3, 125.0,
124.5, 118.0, 116.4, 60.1, 55.2, 43.1, 41.7, 37.8, 32.5, 28.0, 26.3,
23.4, 16.0, 14.1, 11.2. One carbon signal in the aromatic area is
missing. HPLC purity: C18 column >99.9%, Phenyl column 99.5%.
HRMS (M+H+): 603.3199, C34H43N4O6 requires 603.3183. Amino
acid analysis: Nle, 0.99; Ile, 1.01 (84% peptide).
Reaction time: 16 h and 6 h, respectively. Purification by prepara-
tive RP-HPLC gave 7 as a white solid (9.8 mg, 15%). 1H NMR
(CD3OD) d (9:1 mixture of rotamers, major rotamer reported)
7.91 (m, 1H), 7.52 (m, 1H), 7.50 (m, 1H), 7.32 (m, 1H), 7.26–7.20
(m, 3H), 7.03 (m, 2H), 6.71 (m, 2H), 4.63 (m, 1H), 4.47 (d,
J = 15.1 Hz, 1H), 4.43 (d, J = 15.1 Hz, 1H), 3.95–3.91 (m, 3H), 3.73
(app. s, 2H), 2.00–1.84 (m, 2H), 1.80–1.63 (m, 3H), 1.47–1.36 (m,
4H), 0.98 (d, J = 6.4 Hz, 3H), 0.96 (d, J = 6.4 Hz, 3H), 0.95 (t,
J = 7.2 Hz, 3H). 13C NMR (CD3OD) d (major rotamer reported)
175.4, 174.7, 170.1, 168.8, 157.0, 145.2, 139.3, 138.1, 136.5,
134.5, 132.5, 131.8, 131.0, 129.8, 128.64, 128.56, 125.1, 124.6,
118.2, 116.4, 55.2, 54.0, 42.0, 41.9, 41.7, 39.2, 32.5, 28.0, 26.2,
23.41, 23.40, 22.0, 14.1. HPLC purity: C18 column >99.9%, Phenyl
column 99.2%. HRMS (M+H+): 617.3350, C35H45N4O6 requires
617.3339. Amino acid analysis: Nle, 1.01; Leu, 0.99 (81% peptide).
4.3.11. Analogue 11
The analogue was prepared according to the general procedure
using 25b (170 mg, 115
(95.0 mg, 183 mol) and DIEA (63.5
pling followed by Fmoc-Nle-OH (106 mg, 299
(154 mg, 295 mol) and DIEA (102 L, 584 mol) in the second.
l
mol), 20 (84.8 mg, 182
L, 364 mol) in the first cou-
mol), PyBOP
lmol), PyBOP
4.3.8. Analogue 8
l
l
l
The analogue was prepared according to the general procedure
l
using 25a (170 mg, 117
(95.8 mg, 184 mol) and DIEA (64.1
pling followed by Fmoc-Val-OH (99.9 mg, 294
(153 mg, 294 mol) and DIEA (103 L, 589 mol) in the second.
l
mol), 20 (85.7 mg, 184
L, 368 mol) in the first cou-
mol), PyBOP
l
mol), PyBOP
l
l
l
l
l
l
Reaction time: 18 h and 16 h, respectively. Purification by prepara-
tive RP-HPLC gave 11 as a white solid (18.5 mg, 26%). 1H NMR
(CD3OD) d (19:1 mixture of rotamers, major rotamer reported)
7.91 (m, 1H), 7.52 (m, 1H),7.46 (m, 1H), 7.34 (m, 1H), 7.24–7.20
(m, 3H), 7.03 (m, 2H), 6.71 (m, 2H), 4.48 (d, J = 15.2 Hz, 1H), 4.44
(d, J = 15.2 Hz, 1H), 4.40 (d, J = 8.6 Hz, 1H), 3.95–3.92 (m, 3H),
3.76 (d, J = 16.3 Hz, 1H), 3.72 (d, J = 16.3 Hz, 1H), 2.02–1.84 (m,
3H), 1.60 (ddq,J = 13.6, 3.4, 7.6 Hz, 1H), 1.47–1.35 (m, 4H), 1.24
(ddq, J = 13.6, 9.0, 7.4 Hz, 1H), 0.97–0.90 (m, 9H). 13C NMR (CD3OD)
d (major rotamer reported) 175.4, 173.7, 170.1, 168.8, 157.0, 145.3,
139.3, 138.0, 136.6, 134.5, 132.5, 131.8, 131.0, 123.0, 128.7, 128.6,
125.0, 124.5, 118.0, 116.4, 60.1, 55.2, 41.9, 41.7, 39.2, 37.9, 32.5,
28.0, 26.3, 23.4, 16.0, 14.1, 11.2. HPLC purity: C18 column
>99.9%, Phenyl column 99.2%. HRMS (M+H+): 617.3327,
l
l
l
l
Reaction time: 19 h and 18 h, respectively. Purification by prepara-
tive RP-HPLC gave 8 as a white solid (4.0 mg, 6%). 1H NMR (CD3OD)
d (17:3 mixture of rotamers, major rotamer reported) 7.94 (m, 1H),
7.90 (m, 1H), 7.52 (m, 1H), 7.48–7.42 (m, 3H), 7.33 (m, 1H), 7.03
(m, 2H), 6.71 (m, 2H), 4.76 (d, J = 15.4 Hz, 1H), 4.70 (d,
J = 15.3 Hz, 1H), 4.42 (d, J = 8.2 Hz, 1H), 3.93 (app. s, 2H), 3.74 (d,
J = 5.9 Hz, 1H), 2.28 (m, 1H), 1.96 (m, 1H), 1.59 (m, 1H), 1.23 (m,
1H), 1.12 (d, J = 6.9 Hz, 3H), 1.09 (d, J = 6.9 Hz, 3H), 0.96–0.88 (m,
6H). HPLC purity: C18 column 99.1%, Phenyl column 99.3%. HRMS
(M+H+): 589.3029, C33H41N4O6 requires 589.3026. Amino acid
analysis: Val, 1.02; Ile, 0.98 (70% peptide).
C35H45N4O6 requires 617.3339. Amino acid analysis: Nle, 1.03;
4.3.9. Analogue 9
Ile, 0.97 (83% peptide).
The analogue was prepared according to the general procedure
using 25b (170 mg, 115
(94.8 mg, 183 mol) and DIEA (63.5
pling cycle followed by Fmoc-Val-OH (100 mg, 292
(152 mg, 292 mol) and DIEA (102 L, 584 mol) in the second cy-
l
mol), 20 (84.8 mg, 182
L, 364 mol) in the first cou-
mol), PyBOP
l
mol), PyBOP
4.3.12. Analogue 12
The analogue was prepared according to the general procedure
using 25b (200 mg, 148
l
l
l
l
l
mol), 20 (102 mg, 220
lmol), PyBOP
l
l
l
(114 mg, 219 mol) and DIEA (76.0
l
l
L, 436 mol). Reaction time:
l
cle. Reaction time: 18 h and 16 h, respectively. Purification by pre-
parative RP-HPLC gave 9 as a white solid (8.3 mg, 12%). 1H NMR
(CD3OD) d (9:1 mixture of rotamers, major rotamer reported)
7.91 (dd, J = 2.2, 1.6 Hz, 1H), 7.53 (m, 1H), 7.47 (m, 1H), 7.34 (m,
1H), 7.25–7.20 (m, 3H), 7.03 (m, 2H), 6.71 (m, 2H), 4.48 (d,
J = 15.3 Hz, 1H), 4.44 (d, J = 15.3 Hz, 1H), 4.40 (d, J = 8.6 Hz, 1H),
3.93 (app. s, 2H), 3.78–3.70 (m, 3H), 2.28 (m, 1H), 1.97 (m, 1H),
1.60 (m, 1H), 1.23 (m, 1H), 1.11 (d, J = 6.9 Hz, 3H), 1.08 (d,
J = 6.9 Hz, 3H), 0.95–0.90 (m, 6H). 13C NMR (CD3OD) d (major rot-
amer reported) 175.4, 173.7, 170.1, 168.2, 157.0, 145.3, 139.2,
138.0, 136.6, 134.5, 132.4, 131.8, 131.0, 130.0, 128.7, 128.6,
125.1, 124.5, 118.1, 116.4, 60.5, 60.0, 41.9, 41.7, 39.2, 37.9, 31.7,
26.3, 19.0, 17.8, 16.0, 11.2. HPLC purity: C18 column 99.0%, Phenyl
column 99.5%. HRMS (M+H+): 603.3181, C34H43N4O6 requires
603.3183. Amino acid analysis: Val, 1.05; Ile, 0.95 (79% peptide).
18 h. Purification by preparative RP-HPLC gave 12 as a white solid
(6.4 mg, 9%). 1H NMR (CD3OD) d (17:3 mixture of rotamers, major
rotamer reported) 7.40 (m, 1H), 7.34 (m, 1H), 7.29 (m, 1H), 7.25–
7.20 (m, 3H), 7.03 (m, 2H), 7.01 (m, 1H), 6.71 (m, 2H), 4.46 (m,
2H), 4.39 (d, J = 8.5 Hz, 1H), 3.91 (app. s, 2H), 3.76 (d, J = 16.3 Hz,
1H), 3.72 (d, J = 16.3 Hz, 1H), 1.96 (m, 1H), 1.59 (ddq, J = 13.6, 3.4,
7.6 Hz, 1H), 1.23 (m, 1H), 0.96–0.90 (m, 6H). HPLC purity: C18 col-
umn >99.9%, Phenyl column >99.9%. HRMS (M+H+): 504.2509,
C29H34N3O5 requires 504.2498.
4.3.13. Analogue 13
The analogue was prepared according to the general procedure
using 26b (263 mg, 179
lmol), Fmoc-Val-OH (183 mg, 540
lmol),
PyBOP (277 mg, 533 mol) and DIEA (186
l
lL, 1.07 mmol). Reaction
time: 19 h. Purification by preparative RP-HPLC gave 13 as a white
solid (10.8 mg, 12%). 1H NMR (CD3OD) d 7.93 (dd, J = 2.1, 1.6 Hz,
1H), 7.51 (dd, J = 2.1, 1.5 Hz, 1H), 7.49 (m, 1H), 7.37 (m, 1H),
7.29–7.23 (m, 3H), 7.02 (m, 2H), 6.70 (m, 2H), 4.62 (app. s, 2H),
3.92 (app. s, 2H), 3.80 (app. s, 2H), 3.73 (d, J = 5.9 Hz, 1H), 2.26
(dqq, J = 5.9, 6.9, 6.9 Hz, 1H), 1.10 (d, J = 6.9 Hz, 3H), 1.07 (d,
J = 6.9 Hz, 3H). 13C NMR (CD3OD) d 175.7, 169.6, 168.1, 157.0,
145.2, 139.2, 138.3, 136.7, 134.7, 132.5, 131.9, 131.0, 130.0,
128.7, 128.5, 124.9, 124.5, 118.2, 116.4, 60.5, 42.6, 41.8, 39.3,
31.7, 19.0, 17.8. HPLC purity: C18 column 98.2%, Phenyl column
98.1%. HRMS (M+H+): 490.2331, C28H32N3O5 requires 490.2342.
4.3.10. Analogue 10
The analogue was prepared according to the general procedure
using 25a (170 mg, 121
(94.9 mg, 182 mol) and DIEA (63.5
pling followed by Fmoc-Nle-OH (103 mg, 292
(152 mg, 292 mol) and DIEA (102 L, 583 mol) in the second.
l
mol), 20 (84.9 mg, 182
L, 365 mol) in the first cou-
mol), PyBOP
lmol), PyBOP
l
l
l
l
l
l
l
Reaction time: 18 h and 16 h, respectively. Purification by prepara-
tive RP-HPLC gave 10 as a white solid (12.5 mg, 17%). 1H NMR
(CD3OD) d (19:1 mixture of rotamers, major rotamer reported)

6934
H. Andersson et al. / Bioorg. Med. Chem. 16 (2008) 6924–6935
4.3.14. Analogue 14
CHO-K1 cell and transfected HEK293 cell membranes were pre-
Resin 26b (222 mg, 150
lmol) was Fmoc deprotected and
pared as described previously.59 In short, the cells were harvested
with 0.2% EDTA (w/v) (in PBS, pH 7.4) and centrifuged for 5 min at
500g at room temperature. After resuspending in PBS, the number
of cells were counted and washed. The cells were then homoge-
nized in 50 mM Tris–HCl (at pH 7.4) using a Polytron (10 s at max-
imum speed) and Potter homogenizer (30 strokes at 1000 rpm) and
then centrifuged for 30 min (30,000g at 4 °C). The pellet was resus-
pended in 50 mM Tris–HCl, centrifuged (30 min 30,000g at 4 °C)
and the supernatant was removed. The resulting pellets were
stored at ꢂ20 °C until use.
cleaved from the resin according to the general procedure. Purifica-
tion by preparative RP-HPLC gave 14 as a white solid (9.4 mg, 16%).
1H NMR (CD3OD) d 7.56 (m, 1H), 7.44 (dd, J = 2.2, 1.6 Hz, 1H), 7.36
(m, 1H), 7.29–7.23 (m, 3H), 7.09 (dd, J = 2.2, 1.5 Hz, 1H), 7.03 (m,
2H), 6.71 (m, 2H), 4.62 (s, 2H), 3.93 (s, 2H), 3.80 (s, 2H). 13C NMR
(CD3OD) d 175.6, 168.9, 157.1, 146.3, 138.2, 137.4, 134.6, 132.2,
132.0, 131.0, 130.1, 128.8, 128.6, 125.5, 124.8, 118.4, 116.4, 42.6,
41.5, 39.1. One carbon signal in the aromatic area is missing. HPLC
purity: C18 column 99.5%, Phenyl column >99.9%. HRMS (M+H+):
391.1652, C23H23N2O4 requires 391.1658.
4.4.2. Enzyme assay
Determination of the aminopeptidase catalytic activity was
4.3.15. Analogue 15
based on the cleavage of the substrate L L-leucine
-Leu-pNA59 into
The analogue was prepared according to the general procedure
and p-nitroaniline. This latter compound displays a characteristic
light absorption maximum at 405 nm. Pellets, prepared as de-
scribed above, were thawed and resuspended using a Polytron
homogenizer in enzyme assay buffer containing 50 mM Tris–HCl
(pH 7.4), 140 mM NaCl, 0.1% (w/v) bovine serum albumin (BSA)
using 22d (381 mg, 354
(224 mg, 430 mol) and DIEA (150
pling followed by Fmoc-Val-OH (334 mg, 982
(512 mg, 981 mol) and DIEA (342 L, 1.96 mmol) in the second.
l
mol), 20 (191 mg, 411
L, 858 mol) in the first cou-
mol), PyBOP
lmol), PyBOP
l
l
l
l
l
l
Reaction time: 18 h and 19 h, respectively. Purification by prepara-
tive RP-HPLC gave 15 as a white solid (25.3 mg, 16%). 1H NMR
(CD3OD) d 7.92 (dd, J = 2.1, 1.6 Hz, 1H), 7.52 (dd, J = 2.1, 1.5 Hz,
1H), 7.48 (m, 1H), 7.03 (m, 2H), 6.71 (m, 2H), 4.53 (d, J = 6.2 Hz,
1H), 3.92 (app. s, 2H), 3.77 (d, J = 6.0 Hz, 1H), 2.27 (dqq, J = 6.0, 6.9,
6.9 Hz, 1H), 2.02 (m, 1H), 1.61 (ddq, J = 13.6, 4.3, 7.5 Hz, 1H), 1.31
(ddq, J = 13.6, 9.0, 7.4 Hz, 1H), 1.11 (d, J = 6.9 Hz, 3H), 1.08 (d,
J = 6.9 Hz, 3H), 1.02(d, J = 6.8 Hz, 3H), 0.96(m, 3H). 13C NMR(CD3OD)
d 170.2, 168.2, 164.1, 157.0, 145.2, 139.2, 136.7, 132.5, 131.0, 125.1,
124.5, 118.1, 116.4, 60.5, 59.1, 41.7, 38.3, 31.7, 26.6, 19.0, 17.8, 16.1,
11.7. HPLC purity: C18 column 99.0%, Phenyl column 98.7%. HRMS
(M+H+): 456.2505, C25H34N3O5 requires 456.2498.
and 100
ture comprised 50
(1.5 mM) and 50 L enzyme assay buffer alone or with test com-
lM phenylmethylsulfonyl fluoride. The incubation mix-
l
L membrane homogenate, 200 -Leu-pNA
lL L
l
pound. The amount of membrane homogenate corresponded to
4 ꢁ 105 CHO-K1 cells and 1.5 ꢁ 105 transfected HEK293 cells in
each well. Assays were carried out at 37 °C in 96-well plates (Med-
isch Labo Service, Menen, Belgium) and the formation of p-nitroan-
iline was followed by measuring the absorption at 405 nm every
5 min between 10 and 50 min in a Bio-Whittaker ELISA reader.
The enzymatic activities were calculated by linear regression anal-
ysis of the time-wise increase of the absorption.
4.4.3. Radioligand binding assays
4.4. Biochemical evaluation
The binding affinities of the compounds were assessed using
CHO-K1 cell membranes. Membrane pellets were thawed and
resuspended using a Polytron homogenizer in 50 mM Tris–HCl
L-Leucine-p-nitroanilide (L-Leu-pNA) was obtained from Sigma–
Aldrich (Bornem, Belgium). Tyr2 of Ang IV was iodinated as de-
scribed by Lahoutte et al.55 using the Iodogen iodination reagent
from Pierce (Erembodegem, Belgium).55 125I was obtained from
MP Biomedicals (Asse, Belgium). Monoiodinated Ang IV was iso-
lated on a GraceVydac C18 monomeric 120A reverse-phase HPLC
column and stored at ꢂ20 °C in 10 mM KH2PO4, pH 6.5, containing
45% ethanol. All other reagents were of the highest grade commer-
cially available. CHO-K1 cells were kindly obtained from the Pas-
teur Institute (Brussels, Belgium).
(pH 7.4) enzyme assay buffer, including 30 mM EDTA/600 lM
1,10-phenantroline (1,10-Phe). The assays were carried out in
polyethylene 24-well plates (Elscolab, Kruibeke, Belgium). Pre-
incubations were carried out for 40 min at 37 °C in a finale volume
of 250
zyme assay buffer (for total binding), 50
with the compounds (for competition binding assays) or 60
unlabelled Ang IV (for non-specific binding). The binding assay
was initiated by adding 50 L enzyme assay buffer containing
125I]Ang IV and the mixture was further incubated for 30 min at
lL containing 150
lL membrane homogenate, 50
lL en-
L enzyme assay buffer
l
lM
l
[
4.4.1. Cell culture, transient transfection and membrane
preparation
37 °C. Final membrane concentrations were the same as for the en-
zyme assays, final chelator concentrations were 5 mM EDTA and
CHO-K1 and HEK293 cell lines were cultured in 75 and 500 cm2
culture flasks in Dulbecco’s modified essential medium (DMEM)
100 l
M 1,10-Phe, the final [125I]Ang IV concentration was 1 nM.
After incubation, the mixture was vacuum filtered using an Inotech
24-well cell-harvester through GF/B glass fibre filters (Whatman)
pre-soaked in 1% (w/v) BSA. After drying, the radioactivity retained
supplemented with
L-glutamine (2 mM), 2% (v/v) of a stock solu-
tion containing 5000 IU/mL penicillin and 5000
lg/mL streptomy-
cin (Invitrogen, Merelbeke, Belgium), 1% (v/v) of a stock solution
containing non-essential amino acids, 1 mM sodium pyruvate
and 10% (v/v) foetal bovine serum (Invitrogen, Merelbeke, Bel-
gium). The cells were grown in 5% CO2 at 37 °C until confluent.
HEK293 cells were transiently transfected with plasmid DNA,
pCIneo containing the gene of human IRAP (kindly obtained from
Prof. M. Tsujimoto, Laboratoty of Cellular Biochemistry, Saitama,
Japan) or pTEJ456 carrying the complete human aminopeptidase
N cDNA.57 The transient transfection was performed as described
in the filters was measured using a Perkin-Elmer c-counter.
4.4.4. Data analysis
All experiments were performed two times with duplicate
determinations each. The calculation of IC50 values from competi-
tion binding (or enzyme inhibition) experiments was performed by
nonlinear regression analysis using GraphPad Prism 4.0. The equi-
librium dissociation constants (Ki values) of the tested compounds
in the binding and enzyme assays were calculated using the equa-
tion Ki = {IC50/(1+[L]/K)} in which [L] is the concentration of free
radioligand (binding) or free substrate concentration (enzyme as-
say) and K the equilibrium dissociation constant (KD) of [125I]Ang
IV (from saturation binding experiments) or the Michaelis–Menten
constant (Km) for substrate cleavage.60
previously with 8
lL/mL LipofectAMINE (Invitrogen, Merelbeke,
Belgium) and 1
l
g/mL plasmid DNA.58 After transfection, the cells
were cultured for 2 more days. IRAP and AP-N transfected HEK293
cells displayed a 10 and 8 times higher enzyme activity than non-
transfected cells.

H. Andersson et al. / Bioorg. Med. Chem. 16 (2008) 6924–6935
6935
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We thank The Swedish Research Council, the Research Council
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