Potent macrocyclic inhibitors of insulin-regulated aminopeptidase (IRAP) by olefin ring-closing metathesis

Article abstract of DOI:10.1021/jm200036n

Macrocyclic analogues of angiotensin IV (Ang IV, Val¹-Tyr ²-Ile³-His⁴-Pro⁵-Phe⁶) targeting the insulin-regulated aminopeptidase (IRAP) have been designed, synthesized, and evaluated biologically. Replacement of His⁴-Pro ⁵-Phe⁶ by a 2-(aminomethyl)phenylacetic acid (AMPAA) moiety and of Val¹ and Ile³ by amino acids bearing olefinic side chains followed by macrocyclization provided potent IRAP inhibitors. The impact of the ring size and the type (saturated versus unsaturated), configuration, and position of the carbon-carbon bridge was assessed. The ring size generally affects the potency more than the carbon-carbon bond characteristics. Replacing Tyr² by β³hTyr or Phe is accepted, while N-methylation of Tyr ² is deleterious for activity. Removal of the carboxyl group in the C-terminal slightly reduced the potency. Inhibitors 7 (K_i = 4.1 nM) and 19 (K_i = 1.8 nM), both encompassing 14-membered ring systems connected to AMPAA, are 10-fold more potent than Ang IV and are also more selective over aminopeptidase N (AP-N). Both compounds displayed high stability against proteolysis by metallopeptidases.

Full text of DOI:10.1021/jm200036n

ARTICLE
pubs.acs.org/jmc
Potent Macrocyclic Inhibitors of Insulin-Regulated Aminopeptidase
(IRAP) by Olefin Ring-Closing Metathesis
Hanna Andersson,† Heidi Demaegdt,‡ Anders Johnsson,† Georges Vauquelin,‡
Gunnar Lindeberg,† Mathias Hallberg,§ Mꢀatꢀe Erdꢀelyi,||,^ Anders Karlꢀen,† and Anders Hallberg*,†
†Department of Medicinal Chemistry, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden
‡Department of Molecular and Biochemical Pharmacology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium
§Department of Pharmaceutical Biosciences, Uppsala University, Box 591, SE-751 24 Uppsala, Sweden
Department of Chemistry, University of Gothenburg, SE-412 96 Gothenburg, Sweden
^Swedish NMR Centre, University of Gothenburg, Box 465, SE-405 30 Gothenburg, Sweden
S Supporting Information
b
ABSTRACT: Macrocyclic analogues of angiotensin IV (Ang
IV, Val1-Tyr2-Ile3-His4-Pro5-Phe6) targeting the insulin-regu-
lated aminopeptidase (IRAP) have been designed, synthesized,
and evaluated biologically. Replacement of His4-Pro5-Phe6 by a
2-(aminomethyl)phenylacetic acid (AMPAA) moiety and of
Val1 and Ile3 by amino acids bearing olefinic side chains fol-
lowed by macrocyclization provided potent IRAP inhibitors.
The impact of the ring size and the type (saturated versus
unsaturated), configuration, and position of the carbonꢀcarbon bridge was assessed. The ring size generally affects the potency
more than the carbonꢀcarbon bond characteristics. Replacing Tyr2 by β3hTyr or Phe is accepted, while N-methylation of Tyr2 is
deleterious for activity. Removal of the carboxyl group in the C-terminal slightly reduced the potency. Inhibitors 7 (Ki = 4.1 nM) and
19 (Ki = 1.8 nM), both encompassing 14-membered ring systems connected to AMPAA, are 10-fold more potent than Ang IV and
are also more selective over aminopeptidase N (AP-N). Both compounds displayed high stability against proteolysis by
metallopeptidases.
’ INTRODUCTION
to be explored to enable the development of significantly improved
enhancers of cognitive function.
Angiotensin IV (Ang IV, Val1-Tyr2-Ile3-His4-Pro5-Phe6), one
of the bioactive peptides of the reninꢀangiotensin system (RAS), is
known to play an important role in cognitive processes.1ꢀ18 The
hexapeptide improves memory and learning, as has been demon-
strated in a large number of animal studies over the past 20 years
(reviewed by Wright et al.19). Ang IV binds with high affinity to
the insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3),20 an
enzyme localized in areas of the brain associated with memory
and learning.21ꢀ23 IRAP, frequently colocalized with the insulin-
responsive glucose transporter GLUT4,24 is a type II transmem-
brane protein that belongs to the same family of aminopeptidases
as aminopeptidase N (AP-N).25 In recent years, IRAP has emerged
as a promising new target for drug intervention, and selective in-
hibition of this enzyme might provide a new approach in the
treatment of memory dysfunction.26ꢀ28 Furthermore, clinical
studies of the cholinesterase inhibitors and NMDA antagonists
presently used in the treatment of Alzheimer’s disease have been
mostly disappointing.29ꢀ32 Thus, there is a strong demand for
efficient agents, conceivably relying on alternative mechanisms
for the treatment of the cognitive decline associated not only with
Alzheimer’s disease, brain trauma, and cerebral ischemia but also
with other age-related neurological diseases. New avenues have
The first SAR studies on Ang IV analogues targeting the
binding site of Ang IV were reported from the research groups of
Wright and Harding.33ꢀ35 Their continuous work focusing on
developing more druglike Ang IV analogues have led to the
discovery of N-hexanoyl-Tyr-Ile-N0-(5-carbamoylpentyl)amide
(PNB-0408), which is a bloodꢀbrain barrier permeable com-
pound exhibiting cognitive-enhancing activity.19,28,36,37 The ac-
tivity profiles of this modified tripeptide and related analogues in
several animal models of dementia are now being established.28,36
From a β-homoamino acid scan of Ang IV followed by re-
placement of His4 or His4-Pro5 by conformationally constrained
residues, Lukaszuk et al. have developed a number of potent,
selective and stable five- and six-residue analogues of Ang IV,38,39
e.g., 1 (AL-40, Figure 1).38 In 2008, a new druglike series of
chromene-based nonpeptidic IRAP inhibitors, e.g., 2 (HFI-437,
Figure 1), was identified by Albiston et al. by utilizing a
combination of homology modeling of the catalytic domain of
IRAP and in silico screening methodologies.40,41 The performance
Received: January 12, 2011
Published: April 08, 2011
r
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Figure 1. Macrocyclic IRAP inhibitor 7 together with recently identified peptidomimetic (138 and 3ꢀ645) and nonpeptidic (240) inhibitors.
in both spatial working and recognition memory paradigms was
improved after administrating one of the inhibitors to the lateral
ventricles.40 This observation strongly supports the hypothesis that
IRAP provides a relevant enzyme target in the search for new
enhancers of cognition. Recently a study involving molecular dock-
ing and site-directed mutagenesis was published. It was concluded
that Phe544 in IRAP plays an essential role in defining the interaction
of 2 and similar IRAP inhibitors with the catalytic site of the
protease.41
strategies were adopted: cyclization of the Fmoc protected linear
sequence attached to the solid phase (Schemes 1 and 2) or in
solution (Scheme 3).
The initial strategy was to perform RCM with the linear
sequence attached to the resin.58 The stability of the polymer-
bound linkers and reagents is one of the major concerns when
applying microwave heating in solid-phase synthesis. The Wang
and 4-(4-formyl-3-methoxyphenoxy)butyrylaminomethyl (FMPB
AM) resins have been shown to withstand a variety of conditions,
including high temperatures,59 and were thus chosen for the
synthesis of the desired compounds. The Grubbs second gen-
eration catalyst (GII) and HoveydaꢀGrubbs second generation
catalyst (HGII) were evaluated, using 1,2-dichloroethane (DCE)
as solvent and microwave heating at a variety of temperatures for
up to 1 h. From these results the cyclization was performed in
preparative scale using HGII (0.15 equiv) at 150 ꢀC for 5 min and
repeated once after the second addition of catalyst. The resins
were treated with DMSO (30 equiv) in CH2Cl2 overnight for
scavenging of ruthenium contaminants before deprotection and
cleavage.60,61 Complex mixtures of poorly separable compounds
were obtained, and only one compound (7) could be purified at a
sufficient amount. In addition to isomerization before ring closure,
leading to ring-contracted products, double bond migration after
ring closure was observed. Analogue 7 is a result of a one-step
shift of the double bond position toward the backbone. Some
product mixtures were subjected to catalytic hydrogenation to
obtain the corresponding saturated compounds. To allow faster
evaluation and better control over the cyclization step, a solution-
phase approach was adopted. The conditions were re-evaluated,
and the reactions were performed in the presence and absence of
benzoquinones. Electron-deficient benzoquinones such as 2,6-
dichloro-1,4-benzoquinone and tetrafluoro-1,4-benzoquinone
have been found to prevent undesirable isomerization during
RCM.62 Here, both 2,6-dichloro-1,4-benzoquinone and 1,4-
benzoquinone (BQ) were efficient in suppressing double-bond
migration and the formation of ring-contracted products, while
In the search for druglike Ang IV peptidomimetics as efficient
IRAP inhibitors we initiated a program employing an alternative,
substrate-based strategy starting with introduction of steric
constraints.42ꢀ45 Macrocyclization of linear molecules offers
many significant advantages. It reduces the accessible conforma-
tional space and frequently affords ligands with high target affinity
and better metabolic stability, membrane permeability, and se-
lectivity.46 We recently reported that formation of 11- to 13-
membered rings by disulfide cyclizations of Ang IV in both the N-
and C-terminal ends and subsequent structural modifications
relying on conformational analyses provided IRAP inhibitors,
exemplified by 3ꢀ6 (Figure 1).45
We here report that replacement of the chemically and metabo-
lically labile disulfide bonds by carbonꢀcarbon bonds via macro-
cyclization employing the olefin ring-closing metathesis reaction
(RCM) provided potent IRAP inhibitors (e.g., 7, Figure 1).47ꢀ51
The present transformation is well recognized for producing
analogues with altered metabolic stability, biological activity, and
conformational properties.52ꢀ56
’ RESULTS AND DISCUSSION
Chemistry. The compounds presented in Table 1 (7ꢀ10 and
17ꢀ26) were prepared by manual SPPS using the 9-fluorenyl-
methoxycarbonyl (Fmoc) protection strategy,57 followed by
side chain to side chain cyclization between residues 1 and 3
by RCM,47,48,50,51 as outlined in Schemes 1ꢀ3. Two different
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Table 1. Stability and Inhibition Activities of Ang IV and Analogues 7ꢀ10 and 17ꢀ26d
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Table 1. Continued
a [3H]AL-11 competition binding in CHO-K1 cell membranes.76 b Evaluated in an enzyme assay comprising recombinant human IRAP transiently
transfected in HEK293 cells. c Evaluated in an enzyme assay comprising recombinant human AP-N transiently transfected in HEK293 cells.
d SD, standard deviation. N.M., not measurable (>100 μM).
Scheme 1. Synthesis of 7ꢀ9 Using Wang Resina
a Reagents and conditions: (a) (i) 2-(azidomethyl)phenylacetic acid, DIC, DMAP, DMF/CH2Cl2 (1:9), 44 ꢀC, 3 h, (ii) acetic anhydride (5.0 equiv),
pyridine (1.0 equiv), DMF, 0.5 h; (b) (i) Fmoc-Hag-OH, DIC, HOBt, CH2Cl2, (ii) PBu3, room temp, on, (iii) 20% piperidine/DMF; (c) (i) Fmoc-
Tyr(tBu)-OH, HATU, DIEA, DMF, room temp, 2 h, (ii) 20% piperidine/DMF; (d) (2S)-Fmoc-2-amino-6-heptenoic acid, HATU, DIEA, DMF, room
temp, 2 h; (e) (i) HGII, DCE, microwave, 140 ꢀC, 5 min, (ii) HGII, DCE, microwave, 140 ꢀC, 5 min, (iii) DMSO, CH2Cl2, room temp, on, (iv) 20%
piperidine/DMF; (f) trifluoroacetic acid (TFA)/triethylsilane (TES)/H2O (90:5:5), room temp, 2 h; (g) H2, Pd/C, EtOH, room temp, 4 h.
tetrachloro-1,4-benzoquinone was inefficient. Cyclization was
effectively achieved under microwave heating at 120 or 140 ꢀC
for 5 min, using a lower amount of catalyst (0.07 equiv of HGII)
in combination with BQ (0.15 equiv) and with DCE as solvent.
Both the E and Z isomers were obtained and isolated. The E
olefin was consistently the predominant product (>60%).
Generally, the amino acids were single-coupled using 1-[bis(di-
methylamino)methyliumyl]-1H-1,2,3-triazolo[4,5-b]pyridine-3-oxide
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hexafluorophosphate (HATU) and N,N-diisopropylethylamine
(DIEA) in DMF, with reaction times of 2 h for primary amines
and overnight for secondary amines. Completeness of the coupling
reactions was normally assessed by small scale cleavage and LCMS
analysis. The existence of secondary amines was examined using
the chloranil test.63,64 Solid-phase Fmoc deprotection was ac-
complished with 20% piperidine in DMF. The couplingꢀdeprotec-
tioncycle was repeated until the desired amino acid sequence had
been synthesized.
As shown in Scheme 1, the synthesis of 7ꢀ9 started with
N,N0-diisopropylcarbodiimide (DIC) mediated coupling of
2-(azidomethyl)phenylacetic acid to Wang resin. The subse-
quent coupling of homoallylglycine (Hag) was conducted by
addition of tributylphosphine to a mixture of resin bound azide
and activated amino acid in CH2Cl2.42,43,65 The remaining amino
acids were coupled as described above. Purification and separa-
tion of the multiple products obtained upon cleavage from the
resin after RCM and deprotection yielded 7. The remaining
product mixtures were combined and subsequently reduced by
catalytic hydrogenation to yield 8 and 9.
Scheme 2. Synthesis of 10 Using FMPB AM Resina
The synthesis of 10 started with the attachment of the
C-terminal benzylamine (BA) moiety to FMPB AM resin through
reductive amination utilizing NaBH(OAc)3 in 1% AcOH/DMF
(Scheme 2). The reaction was performed under microwave
heating at 60 ꢀC for 20 min.45,66 Standard couplings were used
for the introduction of the three amino acid sequence. The at-
tachment of BA and the initial coupling were monitored by
colorimetric tests using the starting resins as references. The in-
cidence of CHO groups was assessed using the p-anisaldehyde
test,67 and the existence of secondary amines was investigated
using the chloranil test.63,64 The products obtained through on-
resin RCM followed by deprotection and cleavage were purified
and then converted to the corresponding saturated analogues by
catalytic hydrogenation, giving two products separable by RP-
HPLC. One product contained the desired 13-membered ring in
the N-terminal part (10), and the other incorporated the
corresponding ring-contracted 12-membered ring. The latter is
a Reagents and conditions: (a) BA, NaBH(OAc)3, AcOH/DMF (1:99),
microwave, 60 ꢀC, 20 min; (b) (i) Fmoc-Hag-OH, HATU, DIEA, DMF,
room temp, on, (ii) 20% piperidine/DMF; (c) (i) Fmoc-Tyr(tBu)-OH,
3-[bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluo-
rophosphate (HBTU), DIEA, DMF, room temp, 2 h; (d) (i) Fmoc-Hag-
OH, HATU, DIEA, DMF, room temp, 2 h, (ii) 20% piperidine/DMF;
(e) (i) HGII, DCE, microwave, 150 ꢀC, 5 min, (ii) HGII, DCE,
microwave, 150 ꢀC, 5 min, (iii) DMSO, CH2Cl2, room temp, on, (iv)
20% piperidine/DMF; (f) TFA/TES/H2O (90:5:5), room temp, 2 h;
(g) H2, Pd/C, EtOH, room temp, 1.5 h.
Scheme 3. Synthesis of 17ꢀ26 by RCM of the Corresponding Fmoc Protected Linear Intermediates 12ꢀ16, Which Were
Synthesized Using 2-Chlorotrityl Chloride Resin or FMPB AM Resina
a Reagents and conditions: (a) (i) 2-(azidomethyl)phenylacetic acid, DIEA, CH2Cl2, room temp, 4 h, (ii) MeOH, 15 min; (b) (i) PBu3, DMF, (ii) Fmoc-
Hag-OH, HATU, DIEA, DMF, room temp, on, (iii) 20% piperidine/DMF; (c) (i) Fmoc-Hag-OH, DIC, HOBt, CH2Cl2, (ii) PBu3, room temp, on, (iii)
20% piperidine/DMF; (d) (i) Fmoc-Tyr(tBu)-OH, Fmoc-β3hTyr(tBu)-OH, Fmoc-Phe-OH or Fmoc-N-Me-Tyr(tBu)-OH, HATU, DIEA, DMF, room
temp, 2 h, (ii) 20% piperidine/DMF; (e) Fmoc-Hag-OH, (2S)-Fmoc-2-amino-6-heptenoic acid or (2S)-Fmoc-2-amino-7-octenoic acid (11), HATU,
DIEA, DMF, room temp, 2 h for primary amines and overnight for secondary amines; (f) TFA/TES/H2O (90:5:5), room temp, 2 h; (g) HGII, BQ,
DCE, microwave, 120 or 140 ꢀC, 5 min; (h) DBU, 3-mercaptopropyl functionalized silica gel, MeCN/DMF/DMSO, 30 min; (i) BA, NaBH(OAc)3,
AcOH/DMF (1:99), microwave, 60 ꢀC, 20 min or 2 ꢁ 20 min; (j) (i) Fmoc-allylglycine (Fmoc-Alg-OH) or Fmoc-Hag-OH, HATU, DIEA, DMF, room
temp, on, (ii) 20% piperidine/DMF.
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not further discussed, as the structural assignment and evaluation
were complicated by the low yield (<0.4 mg)
proceeds in the absence of chelators, the competition curve will
shift to the right (with respect to the situation in where the com-
pound is exposed to the membranes in the continuous presence
of the chelators), and the compound is quickly degraded by
IRAP, AP-N, and/or other EDTA/1,10-Phe-sensitive metallo-
peptidases. However, the absence of shift does not per se exclude
potential degradation of the compound by alternative routes.
The binding affinities and IRAP activity inhibition data of the
compounds are presented in Table 1.
In the synthesis of 23 and 24, (2S)-Fmoc-2-amino-7-octenoic
acid (11) was required in the N-terminal position in the linear
sequence (Scheme 3). It was obtained by transformation of the
corresponding Boc-protected amino acid, which was commer-
cially available. Removal of the N-Boc group with HCl in 1,4-
dioxane gave the amine hydrochloride salt, which was neutralized
and acylated by Fmoc-Cl under basic conditions (Supporting
Information pp S6ꢀS7).
With a series of disulfide containing Ang IV analogues as starting
points,45 compounds with olefinic carbon bridges of E and Z
configuration and more flexible saturated carbon bridges repla-
cing the disulfide moiety were investigated. Compound 9 (Table 1),
comprising a 13-membered macrocyclic system and a C-terminal
AMPAA group, is only 3 times less efficient as an IRAP inhibitor
(Ki = 64.2 nM) than the parent disulfide analogue 3 (Ki = 22.5 nM,
Figure 1). The selectivity over AP-N is lower (i.e., Ki(AP-N)/
Ki(IRAP)) and the stability somewhat higher as determined from
binding affinity in the presence and absence of metal chelators.
The less flexible E and Z olefin analogues 17 and 18 displayed
similar results, i.e., a 2-fold lower IRAP activity than 3 (Ki = 50.0
and 41.1 nM, respectively), poorer selectivity, and slightly better
stability. As with 9, the C-terminally modified analogue 10 can be
compared to the corresponding disulfide 4. The 2-fold difference
in IRAP activity between 10 (Ki = 193 nM) and 4 (Ki = 95.7 nM)
is in accordance with the results obtained with 9 and 3. The
general trend is that the disulfide bridged analogues are more
selective than the analogues comprising carbonꢀcarbon bridges.
To conclude, the replacement of a disulfide bridge by different
carbonꢀcarbon bridges is well tolerated in 3 and 4, giving analogues
9, 10, 17, and 18, which suggests that there are similarities in
the conformational properties between these 13-membered ring
systems.
The importance of the C-terminal carboxyl group has been ad-
dressed previously (cf. 3 and 4).45 A 5-fold lower IRAP activity
was observed for analogues where AMPAA was replaced by BA.
Similar to the disulfides, a 3 times less potent IRAP inhibitor
(10, Ki = 193 nM) was obtained by the same transformation of 9
(Ki = 64.2 nM). Generally, the γ-turn mimetic moiety AMPAA
shows the best results, but it is evident that structural modifica-
tions are allowed in the C-terminal.
A one-carbon expansion of the 13-membered macrocycle of 9
resulted in a 2-fold potency increase (8, Ki = 25.0 nM). By in-
troduction of a double bond in the resulting 14-membered ring
system, a 6 times more potent inhibitor was obtained (7, Ki =
4.1 nM). Thus, a preference for 14-membered over 13-mem-
bered macrocycles was observed, indicating that the larger rings
can adopt conformations different from those of the smaller rings
upon interaction with IRAP.
The synthesis of analogues 17ꢀ20 (Scheme 3) was initiated
by attachment of 2-(azidomethyl)phenylacetic acid42 to 2-chlor-
otrityl chloride resin. The subsequent coupling of Fmoc-Hag-
OH was conducted by addition of tributylphosphine to a mixture
of resin bound azide and activated amino acid in CH2Cl2. The
remaining amino acids were coupled as described above. The first
two steps in the synthesis of analogues 21ꢀ26 (Scheme 3) were
performed as described for analogue 10 (Scheme 2).
The Fmoc protected linear sequences were cleaved from the
resin, purified by RP-HPLC, and subjected to RCM (12ꢀ16).
The reaction was carried out in DCE under microwave heating at
120 or 140 ꢀC for 5 min using HGII and BQ. Fmoc deprotec-
tion was performed using a few equivalents of 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU) in combination with 3-mercaptopro-
pyl-functionalized silica gel as a dibenzofulvene scavenger.68 To
dissolve the lipophilic Fmoc protected compounds, DMF or
DMSO (5ꢀ15%) was used in addition to CH3CN. The reaction
was completed in minutes, and after filtration and evaporation,
the crude products were dissolved in appropriate mixtures of
DMF/DMSO/CH3CN/H2O (0.1% TFA) and separated using
RP-HPLC.
The double-bond configurations of the final compounds were
established by primitive exclusive correlation spectroscopy (PE-
COSY) and nuclear Overhauser effect spectroscopy (NOESY)
experiments.69ꢀ71 The olefinic 3JHH coupling constants were de-
termined from the corresponding 2D PE-COSY spectra (3JE =
3
15.2ꢀ18.0 Hz, JZ = 8.5ꢀ14.2 Hz, Supporting Information
Figure S1). Where extensive signal overlap of the Z-olefinic protons
made the direct extraction of active couplings impossible, they
were extracted as passive couplings from the cross-peaks to the
neighboring methylene groups. The confirmation of the config-
urational assignment of the E isomers was provided by NOEs
from the methylene protons on either side of the double bond to
the olefinic protons (Supporting Information Figure S2).
Biochemical Evaluation. The ability of 7ꢀ10 and 17ꢀ26 to
inhibit the catalytic activity of recombinant human IRAP and AP-
N transiently transfected in HEK293 cells was examined and
compared. Furthermore, the stability to degradation by metallo-
peptidases in membrane homogenates was investigated for the
majority of the analogues. To this end, membranes of endogen-
ous IRAP-containing CHO-K1 cells were preincubated with
different concentrations of compounds in the absence or pre-
sence of 5 mM 2-[2-[bis(carboxymethyl)amino]ethyl(carboxy-
methyl)amino]acetic acid (EDTA) and 100 μM 1,10-phenantro-
line (1,10-Phe), then further incubated with radioligand in the
presence of both chelators. Because of the presence of the chelators
during the binding step whether or not they were present during
the preincubation, binding affinities solely refer to the IRAP apo-
enzyme.72ꢀ74 In this respect, similar removal of the catalytic zinc
by the synergistic action of both chelators has also been observed
to take place with AP-N, which like IRAP is also part of the M1
family of gluzincin aminopeptidases.75 When the preincubation
Extension of the backbone by the insertion of β-amino acids
influences the secondary structure by altering intramolecular
interactions like hydrogen bonding, which sometimes leads to
enhanced structural stability and β-amino acids acting as turn
mimetics.77ꢀ79 Moreover, the proteolytic stability is frequently
improved.39,80 The Tyr2 residue was previously replaced by
β3hTyr, a modification that rendered potent ligands such as 5
and 6.45 By replacement of the Tyr residue in the 13-membered
analogue 18 (Ki = 41.1 nM), a slightly more potent 14-mem-
bered analogue 20 (Ki = 30.4 nM) was obtained. When the same
transformation was performed with the E isomer 17 (Ki =
50.0 nM), the most potent compound in this and previous series
of Ang IV analogues was obtained, i.e., the 14-membered analogue
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and a lack of proper coordination to the catalytic zinc ion by the
amide nitrogen and possibly also by the oxygen are reasonable
explanations of the outcome of the N-methylation of Tyr2 in the
cyclic Ang IV analogues (Figure 2).83ꢀ86 However, the amide
oxygen has previously been shown not to be essential for high
potency. Thus, Val-Ψ[CH2NH]-Tyr-Val-Ψ[CH2NH]-His-Pro-
Phe (divalinal-Ang IV, Ki = 194 nM), incorporating a reduced
peptide bond between residues 1 and 2 and between residues 3
and 4, is only 4 times less potent as an IRAP inhibitor compared
to Ang IV.73 The configuration of the double bond had a minor
impact on the IRAP activity, while shifting of the double bond
position one step away from the backbone resulted in a 2-fold
decrease (c.f. 23ꢀ26).
To assess the importance of the hydroxyl group, Tyr2 was
replaced by Phe. It is known that [Phe2]Ang IV (Ki of 1.00 nM)
exhibits a slightly better binding affinity than Ang IV (Ki of
2.63 nM) to bovine adrenal membranes, as determined from the
ability to compete with the binding of [125I]Ang IV.33 Its ability
to inhibit IRAP has, to our knowledge, not been determined. In
general, regarding the inhibitors encompassing 13-membered ring
systems, no significant differences were observed when changing
the type of two-carbon bridge. The outcome of 21 and 22 is in
agreement with these observations. No more than a 2-fold dif-
ference was seen in activity between the E and Z isomers. On the
basis of the above discussion, it is anticipated that 10 will be com-
parable to the corresponding unsaturated analogues. Consequently,
since inhibitors 21 (Ki = 697 nM) and 22 (Ki = 351 nM) are
respectively 2 and 4 times less active than 10 (Ki = 193 nM), the
hydroxyl group seems to be beneficial but not a prerequisite for
IRAP activity.
Figure 2. Proposed recognition elements of Ang IV and the Ang IV
analogues (exemplified by 7) important for IRAP interaction. Alterna-
tively, the amide oxygen is coordinated to the catalytic zinc ion.
19 (Ki = 1.8 nM). Notably, while there was no difference in bio-
activity with regard to the E and Z isomers 17 and 18, a significant
difference was seen in the case of the corresponding one-carbon
enlarged E and Z isomers 19 and 20, presumably due to more
favorable conformational properties of 19. Compared to analogue
7 (Ki = 4.1 nM), encompassing a 14-membered macrocycle with a
differently located E olefin element and a Tyr instead of a β3hTyr
residue, 19 is a 2-fold more potent IRAP inhibitor. Both analogues
19 and 7 were more stable than similar disulfides, but only 7
displayed similar selectivity for IRAP over AP-N (i.e., 1000-fold).
The selectivity was 10 times lower for compound 19.
The fact that the macrocylic two-carbon analogues show
improved stability compared to the substrate-like disulfides but
not full stability in the CHO-K1 cells indicates that it is not only
the disulfide bridge that makes the compounds susceptible to
degradation.45 Many reports on improved proteolytic stability,
potency, selectivity, and membrane permeability of peptides and
peptidomimetics have been attributed to mono- and multiple
N-methylation of amino acid residues.77,81,82 It can also result in
significant conformational changes due to altered configurational
properties of the N-methylated peptide bonds bearing a sterically
demanding methyl group, and a reduced number of hydrogen
bond donors preventing intra- and intermolecular interactions.
With the second best IRAP inhibitory effect and the best stability
and selectivity over AP-N, 7 is one of the most promising com-
pounds in the series. In order to investigate the importance of the
NH group in the peptide bond between residues 1 and 2 and to
potentially improve the stability, the Tyr2 residue of the corre-
sponding analogue incorporating BA in the C-terminus was
subjected to N-methylation. This modification was found to have
a significant impact on the activity (cf. 7 and 23). However, part
of the reduced activity is probably attributed to the lack of the
C-terminal carboxyl group in 23 (cf. 9 and 10). Since the
analogue was devoid of all binding affinity, no conclusions could
be drawn concerning its ability to resist degradation by metallo-
peptidases in CHO-K1 cells. A deleterious conformational impact
’ CONCLUSIONS
The design, synthesis, and biochemical evaluation of novel 13-
and 14-membered macrocyclic tripeptide analogues of Ang IV
are presented. It was demonstrated that the replacement of a
disulfide bridge by a carbonꢀcarbon bridge in the N-terminal
macrocyclic part was well tolerated. The IRAP-inhibitory effects
were in the same range as for the disulfides, and the stability to
degradation by metallopeptidases was somewhat improved, while
the selectivity over AP-N was reduced. A preference for 14-
membered rings over 13-membered rings was observed. Gen-
erally, the type (saturated versus unsaturated), configuration, and
position of the carbonꢀcarbon bond did not have a considerable
impact on the outcome. However, 7 (E) was superior to the
saturated analogue 8, and 19 (E) was found to be better than 20
(Z). Hence, the potency generally varied more with the type of
macrocycle than with the specific characteristics of the car-
bonꢀcarbon bond. Substitution of Tyr2 by β3hTyr was well
tolerated. Notably, N-methylation of Tyr2 was deleterious to
activity, conceivably because of the obstruction of coordination
to the catalytic zinc ion or unfavorable conformational properties
of these compounds. The most potent, selective, and stable
analogues in the series, 7 (Ki = 4.1 nM) and 19 (Ki = 1.8 nM),
serve as starting points for further optimization.
’ EXPERIMENTAL SECTION
General Information. Microwave heated reactions were per-
formed in a Smith Synthesizer (Biotage AB, Uppsala, Sweden) produ-
cing controlled irradiation at 2450 MHz. Analytical RP-HPLCꢀMS was
performed on a Gilson HPLC system with a Finnigan AQA quadrupole
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Journal of Medicinal Chemistry
ARTICLE
mass spectrometer using an Onyx monolithic C18 column (4.6 mm ꢁ
50 mm) with gradients of CH3CN/H2O (0.05% HCOOH) or MeOH/
H2O (0.05% HCOOH) at a flow rate of 4 mL/min. Both UV (DAD, 214
and 254 nm) and MS (ESI) detection was utilized. Preparative RP-
HPLC was performed using a Zorbax SB-C8 column (5 μm, 21.2 mm ꢁ
150 mm) with gradients of CH3CN/H2O (0.1% TFA) at a flow rate of
5 mL/min and UV detection at 230 nm. The purity of the compounds
was determined on a Zorbax SB-C8 column (5 μm, 4.6 mm ꢁ 50 mm)
and an Allure biphenyl column (5 μm, 4.6 mm ꢁ 50 mm) using the same
buffer systems at a flow rate of 2 mL/min and with UV detection at
220 nm. A purity of more than 95% was established for all compounds
with the exception of 18 (g92.5%). NMR spectra were recorded on a
Varian Mercury Plus spectrometer (1H at 400 MHz and 13C at 101 MHz)
5ꢀ7 h, MeOH was added, and the mixture was stirred for at least 15 min
before the resin was transferred to a 5 mL disposable syringe fitted with a
porous polyethylene filter. The resin was then washed with several
portions of, in turn, CH2Cl2, DMF, MeOH, and CH2Cl2 and dried in vacuo.
General Procedure D: Coupling to 2-(Azidomethyl)phe-
nylacetyl-2-chlorotrityl Resin Type 1. A round-bottomed flask
was charged with the appropriate amino acid (1.1ꢀ1.5 equiv), HOBt
(1.1ꢀ1.5 equiv), and DIC (1.1ꢀ1.5 equiv) in dry CH2Cl2. After
preactivation under nitrogen for 10 min, the amino acid was treated
with 2-(azidomethyl)phenylacetyl-2-chlorotrityl resin (1.0 equiv), fol-
lowed 10 min later by PBu3 (1.1ꢀ2.0 equiv). The reaction mixture was
stirred under nitrogen for 6 h and then transferred to a 5 mL disposable
syringe fitted with a porous polyethylene filter. The resin was washed
with several portions of, in turn, CH2Cl2, DMF, MeOH, and CH2Cl2
and dried in vacuo.
General Procedure E: Coupling to 2-(Azidomethyl)phe-
nylacetyl-2-chlorotrityl Resin Type 2. 2-(Azidomethyl)phenyl-
acetyl-2-chlorotrityl resin was added to a round-bottomed flask, swollen
in DMF under gentle stirring, treated with PBu3 (1.2 equiv), followed 10
min later by a mixture of the appropriate amino acid (1.2 equiv), HATU
(1.2 equiv), and DIEA (2.4 equiv) in DMF. The reaction mixture was
stirred under nitrogen overnight and then transferred to a 5 mL dis-
posable syringe fitted with a porous polyethylene filter. The resin was
washed with several portions of, in turn, DMF, MeOH, and CH2Cl2 and
dried in vacuo.
General Procedure F: Coupling and Deprotection of Ami-
no Acids. Unless otherwise stated, a solution of the appropriate amino
acid (1.1ꢀ3.0 equiv), HATU (1.1ꢀ3.0 equiv), and DIEA (2.2ꢀ6 equiv)
in DMF was added to the resin and the reaction mixture was agitated by
rotation for 2 h (primary amines) or overnight (secondary amines).
After the resin was washed with several portions of DMF, the resin was
treated with 20% piperidine in DMF (2 ꢁ 4 mL) to remove the Fmoc
group and again rinsed thoroughly with DMF. Upon completion of the
coupling cycle the resin was also washed with several portions of, in turn,
CH2Cl2, MeOH, and CH2Cl2, and dried in vacuo.
General Procedure G: RCM on Resin and Fmoc Deprotec-
tion. The resin was transferred to a Biotage microwave vial (10ꢀ20 mL)
and preswollen in DCE under nitrogen gas. A solution of HGII (0.15
equiv) in DCE was added, and the tube was sealed with a Teflon coated
septa under air. The reaction mixture was heated to 140 or 150 ꢀC by
microwave irradiation for 5 min and then cooled to room temperature.
A needle was used to relieve the pressure in the vial before the addition of
a second portion of HGII (0.15 equiv) in DCE. The reaction mixture
was heated to 140 or 150 ꢀC for another 5 min, after which the resin was
transferred to a 5 mL disposable syringe and washed with several portions
of, in turn, CH2Cl2, DMF, and CH2Cl2. A mixture of DMSO (30 equiv)
and CH2Cl2 was added to the resin. After rotation overnight the resin
was washed with CH2Cl2 and DMF. Fmoc deprotection by treatment
with 20% piperidine in DMF (2 ꢁ 4 mL) was followed by washing with
several portions of, in turn, CH2Cl2, DMF, MeOH, and CH2Cl2 and
drying in vacuo.
and Varian INOVA spectrometers (1H at 900, 800, or 600 MHz and 13
C
at 151 MHz) at ambient temperature. Chemical shifts (δ) are reported
in ppm referenced indirectly to TMS via the 2H lock signal. Assignments
were made using gCOSY, PE-COSY, TOCSY, NOESY, gHSQC, and
gHMBC experiments. Mixing times between 0.4 and 0.6 s were used in
NOESY experiments. PE-COSY spectra were acquired with 4096 ꢁ
2048 data points and zero-filled to 16384 ꢁ 8192 points for measure-
ments of 3JHH. The amino acid residues at positions 1 and 3 are denoted
Xaa1 and Xaa3, respectively. Exact molecular masses were determined on
a Micromass Q-Tof2 mass spectrometer equipped with an electrospray
ion source at the Department of Pharmaceutical Biosciences, Uppsala
University, Sweden. The manual solid-phase synthesis was performed in
disposable syringes fitted with porous polyethylene filters, and a Stuart
SB2 tube rotator was used for agitation. The resins were purchased from
Novabiochem and the unnatural amino acids from RSP Amino Acids or
Sigma-Aldrich. The synthesis of 2-(azidomethyl)phenylacetic acid has
been described earlier.42 Dichloromethane used in the reactions was
distilled over calcium hydride. Other chemicals were purchased and used
without further purification. Final product yields were determined based
on the initial loading of the resin.
General Procedure A: Attachment of 2-(Azidomethyl)phe-
nylacetic Acid to Wang Resin87. Wang resin was added to a Biotage
microwave vial and swollen in CH2Cl2/DMF (9:1) under gentle stirring
for 30 min. A solution of 2-(azidomethyl)phenylacetic acid (2.0 equiv)
and DIC (2.0 equiv) in CH2Cl2/DMF (9:1) was added, followed by
DMAP (0.1 equiv) in CH2Cl2/DMF (9:1). After being sealed, the vial
was flushed with nitrogen gas and the reaction mixture was heated to
44 ꢀC in a heating block for 3 h, after which it was transferred to a 5 mL
disposable syringe fitted with a porous polyethylene filter. The resin was
washed with several portions of, in turn, CH2Cl2 and DMF. The remaining
hydroxyl groups on the resin were capped using acetic anhydride (5.0
equiv) and pyridine (1.0 equiv) in DMF. After 30 min the resin was washed
with several portions of, in turn, DMF, MeOH, and CH2Cl2 and then
used directly in the subsequent step.
General Procedure B: Reductive Amination of FMPB AM
Resin. FMPB AM resin, NaBH(OAc)3 (5ꢀ10 equiv), and DMF were
added to a 10ꢀ20 mL Biotage microwave vial. After being sealed, the vial
was flushed with nitrogen gas, and BA (5ꢀ10 equiv), and AcOH (cat.)
were added. The reaction mixture was heated to 60 ꢀC by microwave
irradiation for 20 min, cooled to room temperature, and then transferred
to a 5 mL disposable syringe fitted with a porous polyethylene filter. The
resin was washed with several portions of, in turn, DMF, MeOH, and
CH2Cl2 and dried in vacuo. The reductive amination and washing were
sometimes repeated once to ensure a sufficient yield. Two colorimetric
tests were employed to examine the reaction, the p-anisaldehyde test for
the presence of CHO groups67 and the chloranil test63,64 for the presence of
secondary amines. The FMPB AM resin was used as a reference.
General Procedure H: Resin Cleavage, Side Chain Depro-
tection, and Purification. The resin was transferred to a 50 mL
Falcon tube containing a stirring bar and treated with a mixture of 95%
TFA (3.0 mL) and TES (0.2 mL) under gentle stirring for 2 h. The resin
was filtered off and further washed with TFA, CH3CN, CH2Cl2, and
MeOH. The filtrate was evaporated, and the residue was dissolved in a
mixture of DMSO/DMF/CH3CN/H2O (0.1% TFA) at appropriate
ratios, filtered through a prerinsed cotton-plugged Pasteur pipet, and
purified using RP-HPLC. Selected fractions were analyzed with HPLCꢀ
MS/UV, and those containing pure product were pooled and lyophilized.
General Procedure I: RCM in Solution, Fmoc Deprotec-
tion, and Purification. The starting material was weighed into a
Biotage microwave vial (2ꢀ5 mL or 10ꢀ20 mL). A stirring bar was
General Procedure C: Attachment of 2-(Azidomethyl)phe-
nylacetic Acid to 2-Chlorotrityl Chloride Resin. 2-(Azidometh-
yl)phenylacetic acid42 (1.0 equiv) was reacted with 2-chlorotrityl chloride
resin (1.5 equiv) in CH2Cl2 in the presence of DIEA (4.0 equiv). After
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Journal of Medicinal Chemistry
ARTICLE
1
added followed by DCE, and HGII (0.07 equiv) and the appropriate
benzoquinone (0.15 equiv) dissolved in DCE (freshly prepared stock
solutions). The tube was sealed with a Teflon coated septa under air. The
reaction mixture was heated to 120 or 140 ꢀC by microwave irradiation
for 5 min and then cooled to room temperature. The reaction mixture
was transferred to a round-bottomed flask, and the solvent was
evaporated. If possible, the crude products were passed through a short
RP column or precipitated in H2O/CH3CN/MeOH and separated by
filtration. The residue was dissolved in DMSO/DMF/CH3CN/H2O
(0.1% TFA), filtered through a prerinsed cotton-plugged Pasteur pipet,
and purified using RP-HPLC. Selected fractions were analyzed using
HPLCꢀMS/UV, and those containing pure product were pooled and
lyophilized. 3-Mercaptopropyl functionalized silica gel (1.2 mmol/g,
5.0ꢀ10 equiv) was weighed into a disposable column, washed thor-
oughly with CH3CN, and then transferred to a glass tube (25 mL). The
purified Fmoc protected compound was dissolved in CH3CN/DMF/
DMSO (ratio adjusted to solubility) and added to the glass tube
followed by DBU (2.5ꢀ5.0 equiv). The tube was sealed with a cap
and rotated on a tube rotator. The reaction was monitored using analytical
HPLCꢀMS/UV. After 30 min the reaction mixture was filtered through
a prerinsed cotton-plugged Pasteur pipet into a round bottomed flask.
The pH of the solution was adjusted to 4 using CH3CN (0.1% TFA),
and the residue remaining after evaporation was diluted with DMSO/
DMF/CH3CN/H2O (0.1% TFA) with appropriate ratios, filtered through
a prerinsed cotton-plugged Pasteur pipet, and purified using RP-HPLC.
Selected fractions were analyzed using HPLCꢀMS/UV, and those
containing pure product were pooled and lyophilized to give the pure
product as the TFA salt.
General Procedure J: Catalytic Hydrogenation of Olefins.
A mixture of the macrocyclic olefins and 10% Pd/C in absolute ethanol
was stirred under hydrogen gas at atmospheric pressure and room
temperature for 1.5ꢀ4 h. After filtration through a disposable syringe filter
and evaporation, the crude product(s) were purified using RP-HPLC.
Compounds 7, 8, and 9. Solid-phase attachment was performed
according to general procedure A. Two batches were made using Wang
resin (402 mg, 261 μmol), 2-(azidomethyl)phenylacetic acid (100 mg,
525 μmol), DIC (81 μL, 520 μmol), DMAP (3.8 mg, 31 μmol), CH2Cl2
(5.4 mL), and DMF (0.6 mL). Capping was achieved using acetic
anhydride (123 μL, 1.31 mmol) and pyridine (21 μL, 261 μmol) in
DMF (3.5 mL). The resin was then reacted with Fmoc-Hag-OH (273 mg,
776 μmol) as described in procedure D using HOBt (106 mg, 783 μmol),
DIC (122 μL, 780 μmol), 85% PBu3 (180 μL, 1.04 mmol), and CH2Cl2
(40 mL). Resin weight: 968 mg. Part of the resin (483 mg, 262 μmol)
was then reacted with Fmoc-Tyr(tBu)-OH (318 mg, 691 μmol) as
described in procedure F using HATU (299 mg, 787 μmol) and DIEA
(273 μL, 1.56 mmol) in DMF (3.0 mL). The same procedure was
applied for the coupling of the N-terminal (2S)-Fmoc-2-amino-6-hepte-
noic acid (208 mg, 347 μmol) residue using HATU (110 mg, 302 μmol),
DIEA (100 μL, 574 μmol), and DMF (3.0 mL). RCM was performed
according to procedure G. The resin was preswollen in DCE (7.0 mL)
for 1 h before the addition of 15 mol % HGII (23.7 mg, 37.8 μmol) in
DCE (2.5 mL). An additional 15 mol % HGII (24.0 mg, 38.3 μmol) in
DCE (2.5 mL) was added before the second round of heating. Workup
using DMSO (543 μL, 7.61 mmol) and CH2Cl2 (3.5 mL) overnight was
followed by Fmoc deprotection. Cleavage from the resin, following
general procedure H, gave a mixture of products, i.e., ring-contracted and/
or isomerized. The only compound separated in good yield by RP-HPLC
was 7, with the double-bond shifted one step toward the backbone. The
other compounds were pooled (4.8 mg, <7.5 μmol) and subjected to
catalytic hydrogenation according to procedure J, using Pd/C (2.5 mg,
2.3 μmol) in absolute ethanol (2.0 mL).
(2.79 mg, 2%). H NMR (400 MHz, 0.1% TFA CD3CN/D2O 4:1)
δ 7.27ꢀ7.18 (m, 3H, AMPAA-ArH), 7.14 (m, 1H, ArH), 7.04 (m, 2H,
Tyr-H2, Tyr-H6), 6.67 (m, 2H, Tyr-H3, Tyr-H5), 5.25 (m, 1H, Xaa1-
Hζ), 5.16 (m, 1H, Xaa3-Hγ), 4.55 (dd, J = 8.3, 6.4 Hz, 1H, Tyr-HR), 4.29
(dd, J = 10.1, 3.5 Hz, 1H, Xaa3-HR), 4.25 (d, J = 15.4 Hz, 1H, AMPAA-
NCH2a), 4.19 (d, J = 15.4 Hz, 1H, AMPAA-NCH2b), 3.81 (dd, J = 9.3,
4.3 Hz, 1H, Xaa1-HR), 3.67 (app s, 2H, AMPAA-CH2CO), 2.92 (dd,
β
β
0
J = 13.7, 8.3 Hz, 1H, Tyr-H ), 2.79 (dd, J = 13.7, 6.4 Hz, 1H, Tyr-H ),
2.40ꢀ2.33 (dm, 1H, Xaa3-Hβ), 2.17 (ddd, J = 14.5, 10.1, 7.9 Hz, 1H,
3
β
1
ε
1
ε
1
β
0
0
Xaa -H ), 1.94 (m, 2H, Xaa -H , Xaa -H ), 1.76 (m, 1H, Xaa -H ),
1
β
0
1.58 (dddd, J = 13.5, 11.8, 9.3, 4.4 Hz, 1H, Xaa -H ), 1.39ꢀ1.25 (m, 2H,
1
δ
1
δ
1
γ
1
γ
0
Xaa -H , Xaa -H ), 1.17ꢀ0.98 (m, 2H, Xaa -H , Xaa -H ). 13C NMR
(101 MHz, 0.1% TFA CD3CN/D2O 4:1) δ 175.3 (AMPAA-CO), 172.5
(Xaa3-CO), 171.8 (Tyr-CO), 170.0 (Xaa1-CO), 156.4 (Tyr-C4), 137.6
(AMPAA-ArC), 134.8 (Xaa1-Cζ), 133.6 (AMPAA-ArC), 131.8 (AMPAA-
ArCH), 131.5 (Tyr-C2, Tyr-C6), 129.0 (AMPAA-ArCH), 128.58
(AMPAA-ArCH), 128.55 (AMPAA-ArCH), 128.47 (Tyr-C1), 127.4
(Xaa3-Cγ), 116.2 (Tyr-C3, Tyr-C5), 56.1 (Tyr-CR), 53.9 (Xaa1-CR),
53.5 (Xaa3-CR), 41.3 (AMPAA-NCH2), 38.8 (AMPAA-CH2CO), 37.2
(Tyr-Cβ), 35.6 (Xaa3-Cβ), 31.6 (Xaa1-Cε), 31.3 (Xaa1-Cβ), 27.5 (Xaa1-
Cδ), 23.0 (Xaa1-Cγ). HPLC purity: C8 column 99.2%, biphenyl column
99.4%. HRMS (M þ Hþ): 537.2718, C29H37N4O6 requires 537.2713.
3JE = 15.6 Hz, measured from PE-COSY spectrum (DMSO-d6).
2-(2-((5S,8S,12S)-5,12-Di(but-3-en-1-yl)-1-(9H-fluoren-9-yl)-
8-(4-hydroxybenzyl)-3,6,10,13-tetraoxo-2-oxa-4,7,11,14-tetra-
azapentadecan-15-yl)phenyl)acetic Acid (13). Solid-phase at-
tachment was done according to general procedure C using 2-(azid-
omethyl)phenylacetic acid (101 mg, 528 μmol), 2-chlorotrityl chloride
resin (544 mg, 767 μmol), DIEA (350 μL, 2.02 mmol), and CH2Cl2
(6.0 mL). MeOH (0.6 mL) was added after 5 h, and the stirring was
continued for 15 min. Resin weight: 612 mg. Part of the resin (604 mg, 520
μmol) was then reacted with Fmoc-Hag-OH (201 mg, 573 μmol) as
described in procedure E using HOBt (77.3 mg, 572 μmol), DIC (81 μL,
517 μmol), 85% PBu3 (99 μL, 571 μmol), and DMF (11 mL). The
subsequent coupling of Fmoc-β3hTyr(tBu)-OH (578 mg, 587 μmol)
was performed with HATU (218 mg, 572 μmol) and DIEA (199 μL,
1.14 mmol) in DMF (3 mL) following general method F. This method
was also applied for the coupling of the N-terminal amino acid Fmoc-
Hag-OH (101 mg, 287 μmol) using HATU (110 mg, 290 μmol) and
DIEA (99.7 μL, 572 μmol) in DMF (3 mL). Cleavage from the resin and
purification, as described in procedure H, gave 13 as a white solid (18.6
mg, 5%). 1H NMR (400 MHz, DMSO-d6) δ 12.34 (br s, 1H, AMPAA-
COOH), 9.12 (s, 1H, β3hTyr-OH), 8.28 (m, 1H, AMPAA-NH), 8.04
(d, J = 7.9 Hz, 1H, Hag3-NH), 7.89 (m, 2H, Fmoc-H4, Fmoc-H5), 7.77
(d, J = 7.8 Hz, 1H, β3hTyr-NH), 7.73 (m, 2H, Fmoc-H1, Fmoc-H8),
7.44 (d, J = 8.4 Hz, Hag1-NH), 7.41 (m, 2H, Fmoc-H3, Fmoc-H6), 7.32
(m, 2H, Fmoc-H2, Fmoc-H7), 7.20ꢀ7.18 (m, 4H, AMPAA-ArH), 6.95
(m, 2H, β3hTyr-H2, β3hTyr-H6), 6.62 (m, 2H, β3hTyr-H3, β3hTyr-
H5), 5.77 (dddd, J = 17.2, 10.0, 6.4, 6.4 Hz, 1H, Hag1-Hδ/Hag3-Hδ),
5.74 (dddd, J = 17.2, 10.0, 6.4, 6.4 Hz, 1H, Hag1-Hδ/Hag3-Hδ), 4.97 (dd,
J = 17.2, 2.0 Hz, 1H, Hag1-Hε/Hag3-Hε), 4.96 (dd, J = 17.2, 1.8 Hz, 1H,
0
1
ε
3
ε
1
ε
3
ε
0
0
Hag -H /Hag -H ), 4.93 (m, 2H, Hag -H , Hag -H ), 4.34ꢀ4.19 (m,
6H, AMPAA-NCH2, Hag3-HR, Fmoc-CH2, Fmoc-H9), 4.17 (m, 1H,
β3hTyr-Hβ), 3.89 (ddd, J = 8.9, 8.4, 5.3 Hz, 1H, Hag1-HR), 3.64 (app s,
2H, AMPAA-CH2CO), 2.63 (dd, J = 13.7, 5.8 Hz, 1H, β3hTyr-Hγ), 2.58
3
γ
0
(dd, J = 13.7, 6.8 Hz, 1H, β hTyr-H ), 2.31 (dd, J = 14.7, 6.6 Hz, 1H,
3
R
3
R
0
β hTyr-H ), 2.25 (dd, J = 14.7, 6.8 Hz, 1H, β hTyr-H ), 2.06ꢀ1.88 (m,
1
γ
1
γ
0
3
γ
3
γ
0
3
β
4H, Hag -H , Hag -H , Hag -H , Hag -H ), 1.74 (m, 1H, Hag -H ),
1.66ꢀ1.49 (m, 3H, Hag -H , Hag -H , Hag -H ). 13C NMR (101
MHz, DMSO-d6) δ 172.5 (AMPAA-CO), 171.6 (Hag3-CO), 171.0
(Hag1-CO), 170.1 (β3hTyr-CO), 155.8 (Fmoc-CO), 155.6 (β3hTyr-
C4), 144.0 (Fmoc-C8a/Fmoc-C9a), 143.8 (Fmoc-C8a/Fmoc-C9a),
140.7 (Fmoc-C4a, Fmoc-C4b), 137.9 (Hag1-Cδ/Hag3-Cδ), 137.8
(Hag1-Cδ/Hag3-Cδ), 137.7 (AMPAA-ArC), 132.9 (AMPAA-ArC),
3
β
1
β
1
β
0
0
2-(2-(((2S,5S,13S,E)-13-Amino-2-(4-hydroxybenzyl)-3,14-
dioxo-1,4-diazacyclotetradec-7-ene-5-carboxamido)methyl)-
phenyl)acetic Acid (7). The product was isolated as the TFA salt
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Journal of Medicinal Chemistry
ARTICLE
130.5 (AMPAA-ArCH), 130.3 (β3hTyr-C2, β3hTyr-C6), 128.4
(β3hTyr-C1), 127.6 (Fmoc-C3, Fmoc-C6), 127.4 (AMPAA-ArCH),
127.1 (Fmoc-C2, Fmoc-C7), 126.8 (AMPAA-ArCH), 126.7 (AMPAA-
ArCH), 125.3 (Fmoc-C1, Fmoc-C8), 120.1 (Fmoc-C4, Fmoc-C5), 115.1
(Hag1-Cε/Hag3-Cε), 115.0 (Hag1-Cε/Hag3-Cε), 114.8 (β3hTyr-C3,
β3hTyr-C5), 65.6 (Fmoc-CH2), 54.3 (Hag1-CR), 52.2 (Hag3-CR),
47.9 (β3hTyr-Cβ), 46.7 (Fmoc-C9), 39.6 (AMPAA-NCH2), 39.3
(β3hTyr-CR), 38.4 (β3hTyr-Cγ), 38.0 (AMPAA-CH2CO), 31.2 (Hag1-
Cβ/Hag3-Cβ), 31.1 (Hag1-Cβ/Hag3-Cβ), 29.26 (Hag1-Cγ/Hag3-Cγ),
29.56 (Hag1-Cγ/Hag3-Cγ), 29.56 (Hag1/Hag3-Cγ). HPLC purity: C8
column 99.6%, biphenyl column 99.2%. HRMS (M þ Hþ): 787.3702,
C46H51N4O8 requires 787.3707.
(β3hTyr-CR), 38.0 (AMPAA-CH2CO), 31.1 (Xaa1-Cβ/Xaa3-Cβ), 30.4
(Xaa3-Cβ/Xaa1-Cβ), 22.6 (Xaa3-Cγ), 21.1 (Xaa1-Cγ). HPLC purity: C8
column 99.0%, biphenyl column 99.4%. HRMS (M þ Hþ): 537.2711,
C29H37N4O6 requires 537.2713.
Compounds 23 and 24. Procedure I was applied for the synthesis
of 23 and 24 starting from 15. RCM was performed using 15 (65.2 mg,
87.8 μmol), HGII (3.51 mg, 5.60 μmol), and BQ (1.42 mg, 13.2 μmol)
in DCE (15 mL). Purification by RP-HPLC (53.9 mg, 75.4 μmol) was
followed by Fmoc deprotection in 10% DMF/MeCN using DBU (2.5
equiv) and 3-mercaptopropyl functionalized silica gel (5.0 equiv). Sep-
aration by RP-HPLC gave the E and Z isomers, 23 and 24, as white
solids.
(2S,5S,13S,E)-13-Amino-N-benzyl-2-(4-hydroxybenzyl)-1-
methyl-3,14-dioxo-1,4-diazacyclotetradec-7-ene-5-carbox-
amide (23). The product was isolated as the TFA salt (31.4 mg, 59%,
16% overall). 1H NMR (600 MHz, DMSO-d6) δ 9.25 (br s, 1H, Tyr-
OH), 8.14 (d, J = 7.9 Hz, 1H, Xaa3-NH), 8.10 (m, 3H, Xaa1-NH3þ), 7.73
(dd, J = 6.5, 5.6 Hz, 1H, BA-NH), 7.31 (m, 2H, BA-H3, BA-H5), 7.24
(m, 1H, BA-H4), 7.19 (m, 2H, BA-H2, BA-H6), 6.99 (m, 2H, Tyr-H2,
Tyr-H6), 6.62 (m, 2H, Tyr-H3, Tyr-H5), 5.28 (m, 1H, Xaa1-Hζ), 5.20
(dd, J = 9.6, 5.1 Hz, 1H, Tyr-HR), 5.15 (m, 1H, Xaa3-Hγ), 4.41 (m, 1H,
Xaa1-HR), 4.28 (dd, J = 15.3, 6.5 Hz, 1H, BA-NCH2a), 4.22 (ddd, J =
11.8, 7.9, 3.5 Hz, 1H, Xaa3-HR), 4.09 (dd, J = 15.3, 5.6 Hz, 1H, BA-
NCH2b), 3.15 (dd, J = 13.6, 9.6 Hz, 1H, Tyr-Hβ), 2.95 (s, 3H, Tyr-
(9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1-(((S)-1-(Benzylami-
no)-1-oxopent-4-en-2-yl)amino)-3-(4-hydroxyphenyl)-1-ox-
opropan-2-yl)(methyl)amino)-1-oxooct-7-en-2-yl)carbamate
(15). Solid-phase attachment was done according to general procedure B
using FMPB AM resin (332 mg, 332 μmol), BA (328 μL, 3.00 mmol),
NaBH(OAc)3 (632 mg, 2.98 mmol), AcOH (0.04 mL), and DMF
(4.50 mL). The reductive amination was repeated once using BA (164
μL, 1.50 mmol), NaBH(OAc)3 (315 mg, 1.49 mmol), AcOH (0.05 mL),
and DMF (8.00 mL). The subsequent couplings were accomplished
following general method F. The resin was reacted with Fmoc-Alg-OH
(125 mg, 370 μmol) using HATU (143 mg, 377 μmol), DIEA (127 μL,
729 μmol), and DMF (3 mL). The coupling of Fmoc-N-Me-Tyr(tBu)-
OH(173 mg, 366 μmol) was performedwith HATU(139 mg, 366 μmol)
and DIEA (127 μL, 729 μmol) in DMF (3.0 mL), and the N-terminal
amino acid (2S)-Fmoc-2-amino-7-octenoic acid (92.7 mg, 244 μmol) was
also introduced by coupling with HATU (109 mg, 287 μmol) and DIEA
(127 μL, 729 μmol) in DMF (3 mL). Cleavage from the resin and
purification, as described in procedure H, gave 15 as a white solid (72.0
mg, 29%). HPLC purity: C8 column 99.0%, biphenyl column 99.0%.
HRMS (M þ Hþ): 743.3807, C45H51N4O6 requires 743.3809.
β
0
3
β
NCH3), 2.50 (m, 1H, Tyr-H ), 2.45 (m, 1H, Xaa -H ), 2.17 (ddd, J =
3
β
1
ε
0
14.4, 11.8, 9.4 Hz, 1H, Xaa -H ), 2.06 (m, 1H, Xaa -H ), 1.88 (m, 1H,
1
ε
1
β
1
β
0
0
Xaa -H ), 1.80 (m, 1H, Xaa -H ), 1.52 (m, 1H, Xaa -H ), 1.34 (m, 1H,
1
δ
1
δ
1
γ
1
γ
0
0
Xaa -H ), 1.27 (m, 2H, Xaa -H , Xaa -H ), 1.08 (m, 1H, Xaa -H ).
3JE = 15.4 Hz, measured from PE-COSY spectrum. 13C NMR (151
MHz, DMSO-d6) δ 171.0 (CO), 168.7 (CO), 168.4 (CO), 155.8 (Tyr-
C4), 139.0 (BA-C1), 133.0 (Xaa1-Cζ), 129.9 (Tyr-C2, Tyr-C6), 128.2
(BA-C3, BA-C5), 127.3 (Tyr-C1), 127.0 (Xaa3-Cγ), 126.9 (BA-C2, BA-
C6), 126.7 (BA-C4), 115.0 (Tyr-C3, Tyr-C5), 57.1 (Tyr-CR), 53.0
(Xaa1-CR), 49.2 (Xaa3-CR), 42.0 (BA-NCH2), 34.4 (Xaa3-Cβ), 33.0
(Tyr-Cβ), 30.6 (Tyr-NCH3), 30.4 (Xaa1-Cε, Xaa1-Cβ), 27.0 (Xaa1-Cδ),
20.8 (Xaa1-Cγ). HPLC purity: C8 column >99.9%, biphenyl column
99.8%. HRMS (M þ Hþ): 493.2804, C28H37N4O4 requires 493.2815.
Biochemical Evaluation. L-Leucine-p-nitroanilide (L-Leu-pNA)
was obtained from Sigma-Aldrich. [3H]AL-11 was obtained from G. Tꢀoth,
Biological Research Center (Szeged, Hungary).76 Allother reagents were of
the highest grade commercially available. CHO-K1 cells were kindly
donated by the Pasteur Institute (Brussels, Belgium).
Compounds 19 and 20. Procedure I was applied for the synthesis
of 19 and 20 starting from 13. RCM was performed using 13 (14.6 mg,
18.6 μmol), HGII (0.73 mg, 1.16 μmol), and BQ (0.27 mg, 2.50 μmol)
in DCE (5.5 mL). The ratios of ring-contracted product/E isomer/Z
isomer were 1:6:2 as determined from HPLCꢀUV. The compounds
were separated by RP-HPLC and subjected to Fmoc deprotection in ap-
proximately 15% DMSO/MeCN using DBU (5 equiv) and 3-mercap-
topropyl functionalized silica gel (10 equiv). Purification by RP-HPLC
gave 19 and 20 as white solids.
2-(2-(((2S,6S,13S,E)-13-Amino-2-(4-hydroxybenzyl)-4,14-
dioxo-1,5-diazacyclotetradec-9-ene-6-carboxamido)meth-
yl)phenyl)acetic Acid (19). The product was isolated as the TFA salt
(3.4 mg, 28%, 1% overall). 1H NMR (600 MHz, DMSO-d6) δ 12.34 (br
s, 1H, AMPAA-COOH), 9.23 (s, 1H, β3hTyr-OH), 8.36 (d, J = 7.7 Hz,
1H, β3hTyr-NH), 8.21 (m, 1H, AMPAA-NH), 8.13 (d, J = 7.6 Hz, 1H,
Xaa3-NH), 8.05 (d, J = 5.6 Hz, 3H, Xaa1-NH3þ), 7.20ꢀ7.17 (m, 4H,
AMPAA-ArH), 7.04 (m, 2H, β3hTyr-H2, β3hTyr-H6), 6.69 (m, 2H,
β3hTyr-H3, β3hTyr-H5), 5.42 (m, 1H, Xaa1-Hδ), 5.33 (m, 1H, Xaa3-
Hδ), 4.22 (m, 2H, AMPAA-NCH2), 4.19 (m, 1H, β3hTyr-Hβ), 3.98 (dd,
J = 14.7, 7.4 Hz, 1H, Xaa3-HR), 3.83 (m, 1H, Xaa1-HR), 3.63 (app s, 2H,
AMPAA-CH2CO), 2.90 (dd, J = 13.5, 5.7 Hz, 1H, β3hTyr-Hγ), 2.73 (dd,
Cell Culture, Transient Transfection, and Membrane Pre-
paration. CHO-K1 and HEK293 cell lines were cultured in 75 and
500 cm2 culture flasks in Dulbecco’s modified essential medium (DMEM)
supplemented with L-glutamine (2 mM), 2% (v/v) of a stock solution
containing 5000 IU/mL penicillin and 5000 μg/mL streptomycin
(Invitrogen, Merelbeke, Belgium), 1% (v/v) of a stock solution contain-
ing nonessential amino acids, 1 mM sodium pyruvate, and 10% (v/v) fetal
bovine serum (Invitrogen, Merelbeke, Belgium). The cells were grown
in 5% CO2 at 37 ꢀC until confluent.
HEK293 cells were transiently transfected with plasmid DNA, with
pCIneo containing the gene of human IRAP (kindly provided by Prof.
M. Tsujimoto, Laboratory of Cellular Biochemistry, Saitama, Japan) or
pTEJ488 carrying the complete human AP-N cDNA.25 The transient
transfection was performed as described previously with 8 μL/mL
Lipofectamine (Invitrogen, Merelbeke, Belgium) and 1 μg/mL plasmid
DNA.89 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, respectively, than nontransfected cells.
3
γ
0
J = 13.8, 7.7 Hz, 1H, β hTyr-H ), 2.70 (dd, J = 14.8, 5.0 Hz, 1H,
β3hTyr-HR), 2.15 (m, 1H, Xaa3-Hγ), 2.10 (dd, J = 14.8, 8.1 Hz, 1H,
3
R
1
γ
1
γ
3
γ
0
0
0
β hTyr-H ), 1.91ꢀ1.79 (m, 3H, Xaa -H , Xaa -H , Xaa -H ),
1
β
1
β
3
β
3
β
0
3
0
1.72ꢀ1.61 (m, 4H, Xaa -H , Xaa -H , Xaa -H , Xaa -H ). JE = 17.1 Hz,
measured from PE-COSY spectrum. 13C NMR (151 MHz, DMSO-d6)
δ 172.5 (CO), 172.2 (CO), 170.7 (CO), 167.6 (CO), 155.9 (β3hTyr-C4),
137.8 (AMPAA-ArC), 132.9 (AMPAA-ArC), 130.4 (β3hTyr-C2, β3hTyr-
C6, AMPAA-ArCH), 129.5 (Xaa1-Cδ), 129.4 (Xaa3-Cδ), 128.2 (β3hTyr-
C1), 127.2 (AMPAA-ArCH), 126.7 (AMPAA-ArCH), 126.6 (AMPAA-
ArCH), 115.0 (β3hTyr-C3, β3hTyr-C5), 51.6 (Xaa1-CR), 51.4 (Xaa3-CR),
49.2 (β3hTyr-Cβ), 39.7 (β3hTyr-Cγ), 39.6 (AMPAA-NCH2), 38.2
CHO-K1 cell and transfected HEK293 cell membranes were pre-
pared as described previously.90 Briefly, the cells were harvested with
0.2% EDTA (w/v) (in phosphate buffered saline (PBS), pH 7.4) and
centrifuged for 5 min at 500g at room temperature. After resuspension in
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dx.doi.org/10.1021/jm200036n |J. Med. Chem. 2011, 54, 3779–3792

Journal of Medicinal Chemistry
ARTICLE
PBS, the cells were counted and washed. The cells were then homo-
genized in 50 mM Tris-HCl (at pH 7.4) using a Polytron homogenizer
(10 s at maximum speed) and a Potter homogenizer (30 strokes at
1000 rpm) and then centrifuged for 30 min (30000g at 4 ꢀC). The pellet
was resuspended in 50 mM Tris-HCl and centrifuged (30 min 30000g
at 4 ꢀC), and the supernatant was removed. The resulting pellets were
stored at ꢀ20 ꢀC until use.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ46 18 4714284. Fax: þ46 18 4714474. E-mail: Anders.
Hallberg@orgfarm.uu.se.
’ ACKNOWLEDGMENT
Enzyme Assay. Determination of the aminopeptidase catalytic
activity was based on the cleavage of the substrate L-leucine-p-nitroani-
lide (L-Leu-pNA)90 to give L-leucine and p-nitroaniline. This latter com-
pound displays a characteristic light absorption maximum at 405 nm.
Pellets, prepared as described 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), and 100 μM phenylmethylsulfonyl fluoride (PMSF). The
incubation mixture comprised 50 μL of membrane homogenate,
200 μL of L-Leu-pNA (1.5 mM), and 50 μL of enzyme assay buffer
alone or with the test compound. The amount of membrane homo-
genate corresponded to 1.5 ꢁ 105 transfected HEK293 cells in each well.
Assayswere carried out at 37 ꢀC in 96-well plates (Medisch Labo Service,
Menen, Belgium), and the formation of p-nitroaniline was monitored by
measuring the absorption at 405 nm every 5 min between 10 and 50 min
in a Tecan M200 96-well reader. The enzymatic activities were calculated by
linear regression analysis of the timewise increase in absorption.
Stability Experiments. The stability of the compounds was
studied in the presence of CHO-K1 cell membranes. Membrane pellets
were thawed and resuspended using a Polytron homogenizer in 50 mM
Tris-HCl (pH 7.4) enzyme assay buffer. Preincubations were carried out
for 40 min at 37 ꢀC in a final volume of 250 μL containing 150 μL of
membrane homogenate (corresponding to 4 ꢁ 105 CHO-K1 cells,
50 μL of enzyme assay buffer without or with 30 mM EDTA/600 μM,
1,10-Phe, and 50 μL of enzyme assay buffer without or with the different
compounds or unlabeled Ang IV (60 μM for nonspecific binding). Then
the binding assay was initiated by adding 50 μL of enzyme assay buffer
containing [3H]AL-11 (18 nM, without (if chelators were already
present in the preincubation medium) or with 30 mM EDTA/600 μM
1,10-Phe), and the mixture was further incubated for 30 min at 37 ꢀC.
The final chelator concentrations were 5 mM EDTA and 100 μM 1,10-
Phe. The [3H]AL-11 concentration was 3 nM, and the final unlabeled
ligand concentrations ranged from 10ꢀ5 to 10ꢀ9 M. After incubation, the
mixture was vacuum-filtered using an Inotech 24-well cell harvester
through GF/B glass fiber filters (Whatman) presoaked in 1% (w/v)
BSA. After the filters were dried, the radioactivity retained by the filters
was measured (after adding 3 mL of scintillation liquid (Optiphase Hisafe))
using a β-counter (Perkin-Elmer). [3H]AL-11 was characterized as de-
scribed by Demaegdt et al.76
We gratefully acknowledge The Swedish Research Council for
economic support. H.D. was a postdoctoral researcher of the
“Fund for Scientific Research—Flanders” (FWO, Belgium). We
are grateful to Prof. D. Tourwꢀe and A. Lukaszuk (Department of
Organic Chemistry, Vrije Universiteit Brussel, Belgium) for
the preparation of the AL-11 precursor and to G. Tꢀoth and
E. Szemenyei (Biological Research Center, Szeged, Hungary) for
the tritiation of this precursor to [3H]AL-11.76 We also thank
Aleh Yahorau for conducting the HRMS experiments.
’ ABBREVIATIONS USED
Alg, allylglycine, (2S)-2-amino-4-pentenoic acid; AMPAA, 2-(amino-
methyl)phenylacetic acid; Ang IV, angiotensin IV; AP-N, amino-
peptidase N; BA, benzylamine; BQ, 1,4-benzoquinone; DBU,
1,8-diazabicyclo[5.4.0]undec-7-ene; DCE, 1,2-dichloroethane;
DIC, N,N0-diisopropylcarbodiimide; DIEA, N,N-diisopropy-
lethylamine; EDTA, 2-[2-[bis(carboxymethyl)amino]ethyl(car-
boxymethyl)amino]acetic acid;Fmoc, 9-fluorenylmethoxycarbonyl;
FMPB AM, 4-(4-formyl-3-methoxyphenoxy)butyrylaminomethyl;
GII, Grubbs catalyst second generation, benzylidene[1,3-bis-
(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclo-
hexylphosphine)ruthenium; Hag, homoallylglycine, (2S)-2-ami-
no-5-hexenoic acid; HATU, 1-[bis(dimethylamino)methylium-
yl]-1H-1,2,3-triazolo[4,5-b]pyridine-3-oxide hexafluorophosphate;
HBTU, 3-[bis(dimethylamino)methyliumyl]-3H-benzotriazol-
1-oxide hexafluorophosphate; HGII, HoveydaꢀGrubbs catalyst
second generation, (1,3-bis-(2,4,6-trimethylphenyl)-2-imidazo-
lidinylidene)dichloro(o-isopropoxyphenylmethylene)ruthenium;
HOBt, 1-hydroxybenzotriazole; IRAP, insulin-regulated amino-
peptidase; NOESY, nuclear Overhauser effect spectroscopy; PE-
COSY, primitive exclusive correlation spectroscopy; RCM, ring-
closing metathesis; RAS, reninꢀangiotensin system; TES, tri-
ethylsilane; TFA, trifluoroacetic acid; Xaa1, amino acid residue
at position 1; Xaa3, amino acid residue at position 3
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pounds in the binding and enzyme assays were calculated using the
equation Ki = [IC50/(1 þ [L]/K)] in which [L] is the concentration of
the free radioligand (binding) or free substrate concentration (enzyme
assay) and K the equilibrium dissociation constant (KD) of [3H]AL-11
(from saturation binding experiments) or the MichaelisꢀMenten con-
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