Angiotensin II analogues encompassing 5,9- and 5,10-fused thiazabicycloalkane tripeptide mimetics

Article abstract of DOI:10.1021/jm991089q

A simple experimental procedure on solid phase for the construction of new tripeptidic 5,9- and 5,10-fused thiazabicycloalkane scaffolds that adopt β-turns has been developed. This N-terminal-directed bicyclization, relying on masked aldehyde precursors d

Full text of DOI:10.1021/jm991089q

4524
J . Med. Chem. 1999, 42, 4524-4537
An gioten sin II An a logu es En com p a ssin g 5,9- a n d 5,10-F u sed
Th ia za bicycloa lk a n e Tr ip ep tid e Mim etics
Petra J ohannesson,† Gunnar Lindeberg,† Weimin Tong,† Adolf Gogoll,‡ Barbro Synnergren,§ Fred Nyberg,§
Anders Karle´n,† and Anders Hallberg*,†
Department of Organic Pharmaceutical Chemistry, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden,
Department of Organic Chemistry, Uppsala University, Box 531, SE-751 21 Uppsala, Sweden, and
Department of Biological Research on Drug Dependence, Uppsala University, Box 591, SE-751 24 Uppsala, Sweden
Received May 31, 1999
A simple experimental procedure on solid phase for the construction of new tripeptidic 5,9-
and 5,10-fused thiazabicycloalkane scaffolds that adopt â-turns has been developed. This
N-terminal-directed bicyclization, relying on masked aldehyde precursors derived from glutamic
acid as key building blocks, provides a complement to the related bicyclization previously
reported, where an aspartic acid-derived precursor was employed to induce cyclization toward
the C-terminal end of the peptide. Thus, the regioselectivity of the bicyclization can be altered
simply by varying the chain length of the incorporated aldehyde precursor. Four analogues of
the hypertensive octapeptide angiotensin II, comprising the new scaffolds in the 3-5- and 5-7-
positions, were synthesized. One of these conformationally constrained angiotensin II analogues
exhibited AT1 receptor affinity (Ki ) 750 nM). Results from theoretical conformational analysis
of model compounds of the bicyclic tripeptide mimetics are presented, and they demonstrate
that subtle differences in geometry have a strong impact on the affinity to the AT1 receptor.
In tr od u ction
Nikiforovich and Marshall to postulate a model of the
bioactive conformation of Ang II when binding to the
AT1 receptor.16,17 Further modifications, now in the 5-7
region of Ang II, provided the bicyclic analogue c[Sar1,
Hcy,5 Mpc7]Ang II, 4, which also has good binding
affinity toward AT1 receptors and exerts a partial
agonistic effect.14 Our incorporation of γ-turn mimetics
into the 3-5 region of Ang II required considerable
synthetic efforts and resulted in an agonist, although
300-fold less potent than Ang II.15
There is a real need for new, complementary, flexible,
and synthetically simple cyclization methods for pep-
tides, which enforce conformational constraints and
allow for fine-tuning of models of bioactive conforma-
tions. These methods should preferably be exploited
prior to the initiation of the often tedious synthetic
programs aimed at more complex organic scaffolds.
Although several methods for the synthesis of con-
strained bicyclic dipeptides, including motifs mimicking
â-turns, have appeared in the literature,18-23 reports on
the corresponding bicyclic tripeptides with reverse turn-
inducing properties are rare.
We have an interest in developing cyclization strate-
gies that stabilize secondary structures, such as â-turns
and γ-turns, in peptides. Previously, we have reported
a procedure on solid phase for the preparation of a series
of Ang II analogues encompassing the bicyclic tripeptide
scaffolds 5 and 6 which have â-turn-inducing properties
(Chart 2).24 A masked ω-formyl R-amino acid derived
from L-aspartic acid was used as building block, and a
stereoselective C-terminal-directed bicyclization was
achieved. The simplicity of the experimental procedure
and the obvious potential of the bicyclization strategy
for the construction of a diversity of constrained back-
bone scaffolds encouraged us to survey its applicability.
We herein report that by employing building blocks
Determination of the bioactive conformations of bio-
logically active peptides remains an exciting challenge.
Linear peptides interconvert rapidly in solution between
conformations which differ little in energy, and knowl-
edge of a preferred solution conformation does not
necessarily provide information regarding the receptor-
bound conformation of the peptide. Insights into the
topological requirements within a peptide-receptor
complex are of the utmost importance in drug discovery
programs, where often the conversion of peptides into
nonpeptidic organic molecules, with high bioavailability
and with retained biological activity, is the ultimate
goal. To date, no three-dimensional structural data are
available for peptides interacting with their G-protein-
coupled receptor targets, and critical information of the
receptor-bound conformation has to be gained in an
indirect fashion. One strategy involves the introduction
of conformational constraints, for example, by cycliza-
tion1,2 or by incorporation of secondary structure
mimetics3-7 at selected sites of the peptide, to enforce
important recognition elements into the correct regions
of space.
This strategy has been successful in the search for
the bioactive conformation of the hypertensive octapep-
tide angiotensin II (Ang II), 1 (Chart 1).8-15 Spear et
al.9 have reported that displacement of Val3 and Ile5 in
Ang II by Hcy3 and Hcy5 and subsequent oxidation
furnished the potent agonist c[Hcy3,5]Ang II, 2. Confor-
mational analysis and further synthetic elaborations
involving the synthesis of the more constrained bicyclic
agonist c[Sar1, Hcy3, Mpt5]Ang II, 3 (Chart 1),11 allowed
† Department of Organic Pharmaceutical Chemistry.
‡ Department of Organic Chemistry.
§ Department of Biological Research on Drug Dependence.
10.1021/jm991089q CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/14/1999

Angiotensin II Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4525
Ch a r t 1
Ch a r t 2
Sch em e 1a
a
Reagents: (a) MeNHOMe‚HCl, NEt3, PyBOP, CH2Cl2, 92%;
(b) DIBALH, THF, 89%; (c) (i) TMS-I, CH2Cl2, (ii) MeOH, CH2Cl2,
(iii) Fmoc-Cl, Na2CO3, H2O/dioxane, 41%.
derived from L-glutamic acid as a substitute for L-
aspartic acid, N-terminal-directed bicyclization can be
accomplished providing the new tripeptidic scaffolds 7a ,
7b, and 8 (Chart 2). We also report the binding affinities
of four Ang II analogues encompassing the new scaffolds
in the 3-5- and the 5-7-positions. Finally, we present
the results from a theoretical conformational analysis
of these scaffolds.
amide.26 Thus, commercially available Boc-Glu-OtBu (9)
was transformed into amide 10 and reduced with
diisobutylaluminum hydride (DIBALH) essentially by
following the procedure of Wernic et al.27 for the
synthesis of the corresponding L-aspartic acid deriva-
tives. The aldehyde formed in this way spontaneously
cyclized to give the 5-hydroxyproline derivative 11 as a
pair of diastereoisomers (approximate ratio 1:1). No
attempts to separate the diastereoisomers were made.
NMR spectra recorded at 20 °C (DMSO-d6) showed each
diastereoisomer to exist as a mixture of two rotamers,
attributable to the relatively high rotation barrier of the
nitrogen-carbon bond of the carbamate function.28,29
Variable temperature NMR confirmed the existence of
a rotamer mixture. The Boc and tert-butyl groups were
removed by trimethylsilyl iodide (TMS-I), and the
zwitterion formed was directly converted to Fmoc
derivative 12. Also for compound 12, each diastereoi-
somer was shown by NMR to exist as a mixture of
rotamers.
Resu lts
Syn th esis. Two alternative Fmoc-protected building
blocks were prepared and thereafter incorporated into
Ang II. The synthesis of the first, Fmoc-protected
5-hydroxyproline 12, is outlined in Scheme 1. The
N-Boc-protected aldehyde derived from glutamic acid
has been reported to spontaneously cyclize to 5-hydroxy-
proline.25 Initial experiments to prepare the aldehyde
by controlled oxidation of the corresponding primary
alcohol were unsuccessful and led to overoxidation to
pyroglutamic acid.25 The aldehyde therefore was pre-
pared under reductive conditions via the Weinreb

4526 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
J ohannesson et al.
Sch em e 2a
and 20 was accompanied with negligible racemization
and that the bicyclizations occur with high regioselec-
tivity. After purification by RP-HPLC, the Ang II
analogues 24-26 were isolated in 55-33% yield.
Str u ctu r a l Deter m in a tion . Proton NMR signals of
Ang II analogues 24-26 (Table 1) were assigned from
P.E.COSY,33 TOCSY,34 and ROESY35 spectra, following
standard methodology.24,36
Absolute configurations at C-δ of Aop were deter-
mined as follows. For 26, S configuration at this center
is revealed by NOEs between the proton at Aop-δ and
Tyr-NH; the position of the latter with respect to the
faces of the bicyclic ring system is determined by its
NOE with the proton at Hcy-R. Notably, no NOE is
observed between the protons at Aop-δ and Tyr-R. This
is in contrast with the observation of a strong NOE
between Aop-δ and Tyr-R in 24a , which is only possible
if the configuration at C-δ of Aop is R. For compound
25, the R configuration at Aop-δ is established with
detection of a NOE between the protons at Aop-δ and
His-R. Finally, in 24b, S configuration at C-δ of Aop is
indicated, similarly as described for 26, by a NOE
between Aop-δ and Tyr-NH. As before, the position of
Tyr-NH was revealed by its NOE with Hcy-R, and the
absence of a NOE between Aop-δ and Tyr-R lends
additional support to this assignment.
a
Reagents: (a) paraformaldehyde, TsOH, benzene, 98%; (b)
It is notable that the chemical shift for the Aop-δ is
correlated to the absolute configuration at this carbon,
i.e., higher chemical shifts are observed for the two
S-configured centers (24b: δ ) 5.34, 26: δ ) 5.52) than
for the R-configured centers (24a : δ ) 4.64, 25: δ )
4.60). This can be compared to the R-configured centers
in the previously reported compounds,24 where the
chemical shifts ranged between δ ) 4.79 and 4.93.
The following observations, which might be related
to solution conformations of Ang II analogues 24-26,
were made. For 24a we note that Tyr-NH has a large
coupling constant (J ) 10.3 Hz) (Table 2), while all other
parameters have average values. In 24b, low chemical
shifts are observed for the NH protons of His (δ ) 7.29)
and Phe (δ ) 8.02), and low temperature coefficients
(Table 3) are observed for His-NH (0.3) and Cys-NH
(2.7). Furthermore, values for the NH coupling con-
stants of Tyr (J ) 4.6 Hz) and Cys (J ) 6.3 Hz) are lower
than average. Analogue 25 shows unusually high chemi-
cal shifts for His protons (δ NH ) 9.15, δ H-R ) 5.06),
as well as a large coupling constant between them (J )
10.2 Hz). Cys-NH has a low coupling constant (J ) 6.9
Hz). Temperature coefficients are moderate to high (7.0
for Cys-NH). In 26, only moderate parameter values are
observed. Tyr-NH has a low temperature coefficient of
1.7.
Me2S‚BH3, THF; (c) PCC, NaHCO3, Celite, CH2Cl2; (d) TsOH,
MeOH, 50% from 14; (e) NaOMe, MeOH, 89%; (f) H2, Pd/C, EtOH,
83%, (g) (i) KOH(aq), MeOH, (ii) Fmoc-Cl, Na2CO3, H2O/dioxane,
62%.
For the synthesis of the second building block 20, as
is depicted in Scheme 2, a protected uncyclized alde-
hyde, 2-amino-5-oxopentanoic acid (Aop), is employed
as a key intermediate. The nitrogen has to be dipro-
tected in order to avoid the facile cyclization onto the
aldehyde function. Thus compound 16, where Aop is
protected as a N-(carbobenzyloxy)oxazolidinone,30 was
synthesized as described previously.31 The formyl group
was thereafter protected as the dimethyl acetal, and the
resulting compound (17) was converted to the free amine
19 via 18.32 After hydrolysis of the R-methyl ester and
subsequent Fmoc protection of the nitrogen, building
block 20 was isolated.
The building blocks 12 and 20 were subjected to solid-
phase peptide synthesis (SPPS) and incorporated into
position 5 of the dimercapto peptide precursors 21 and
22, respectively (Scheme 3). Upon deprotection and
cleavage from the solid phase with TFA, both peptides
bicyclized toward the N-terminal end to deliver the Ang
II analogues 23a and 23b, comprising the 5,9-bicyclic
tripeptide units 7a and 7b.
Building block 20 was found to give more easily
purified bicyclized peptides than building block 12 and
was therefore selected for the synthesis of Ang II
analogues 24-26 (Scheme 4). The bicyclization deliv-
ered one major diastereoisomer except in the case of
compounds 24a and 24b, where the two ring junction
epimers were both isolated. Analogues 24-26 were
formed as the predominant components of the reaction
mixtures. According to LC-MS analysis none of the
major side products (>5% yields as estimated from peak
areas) exhibited the same molecular weights as the
isolated products, suggesting that the synthesis of 12
In Vitr o Bin d in g Affin ity. Compounds 24-26 were
screened in a radioligand binding assay based on the
displacement of [125I]Ang II from rat AT1 receptors
stably expressed by Chinese hamster ovary (CHO)
cells.37,38 Compounds exhibiting affinity (i.e., compound
26) were further evaluated in a second radioligand
binding assay on AT1 receptors in rat liver mem-
branes11,39 in order to obtain more precise Ki values.
Ang II analogue 26 displayed an affinity with a Ki value
of 750 nM in this assay. The results from the two
receptor binding assays are shown in Table 4. Ang II,

Angiotensin II Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4527
Sch em e 3a
a
Treatment of the dimercapto precursors 21 and 22 with TFA delivered the N-terminal-bicyclized Ang II analogues 23a and 23b.
c[Hcy3,5]Ang II, 2, and the nonpeptide AT1 antagonist
DuP 753 were used as reference substances in both
assays.
plot in Figure 2a, these distances are plotted for all
conformations within 5 kcal/mol of the lowest-energy
conformation. Only 7a and 28 adopt â-turns as defined
by the CR1-CR4 distance. It should be noted that this
turn cannot be stabilized by an intramolecular hydrogen
bond in 7a , since the nitrogen of residue 4 lacks a
hydrogen atom. Model compounds 7b and 8 only adopt
â-turns as defined by the CR2-CR5 distance. Nine out
of the 21 conformations of 7b and 19 out of the 44
conformations of 8 adopt these â-turns. Five of the
â-turn conformations of 7b and six of the â-turn
conformations of 8 correspond to the type I â-turn.44 In
Figure 1, a conformation of 7b adopting a type I â-turn
is shown. Since a proline derivative is present in
position 3 in the â-turn between CR2-CR5, we also
searched for type VI â-turns.44,45 However, this turn
(type VIa) was only found in one of the conformations
of 8.
Th eor etica l Con for m a tion a l Ch a r a cter iza tion of
Sca ffold s 7a , 7b, a n d 8. Conformational analysis was
performed on the model compounds of scaffolds 7a , 7b,
8, and 27 (See Charts 2 and 3). The conformational
properties of the disulfide scaffold 27, incorporated in
the potent Ang II agonist 3 (Chart 1), have been
reported previously.11,40 We reanalyzed 27 to allow for
a direct comparison with the model compounds of our
scaffolds (see Discussion). We also included the blocked
Ala-Ala-Ala tripeptide 28 in the analyses, as a linear
reference peptide. The Amber* force field and the GB/
SA water solvation model41 within Macromodel (version
5.5)42 were used in the calculations, and all conforma-
tions within 5 kcal/mol of the lowest-energy minimum
were characterized. The number of conformations within
5 kcal/mol of the global energy minimum found for
model compounds 7a , 7b, 8, 27, and 28 was 14, 21, 44,
58, and 24, respectively.
To characterize model compounds 7a , 7b, and 8, we
studied their â-turn-inducing propensities by measuring
the distance between CR of the first residue to CR of the
fourth residue. In a â-turn, by definition, this distance
should be less than 7 Å and the tetrapeptide sequence
making up the â-turn should not be part of an R-helical
region.43 For the model compounds used in this study,
two locations of the â-turn are available, either between
residues 1 and 4 (CR1-CR4 < 7 Å) or between residues
2 and 5 (CR2-CR5 < 7 Å, see Figure 1). In the scatter
The conformational preferences of the backbone tor-
sion angles that are restricted within the bicyclic moiety
of 7a , 7b, and 8 (Ψ2, Φ3, Ψ3, and Φ4; see Figure 1) were
also studied. In Figure 2b the Φ3,Ψ3 plot and in Figure
2c the Φ4,Ψ2 plot of all conformations within 5 kcal/mol
of the lowest-energy conformation are shown. In Figure
2b it can be seen that the stereochemically identical
compounds 7b and 8 have similar torsion angle values.
In the region Φ3 ) -60° and Ψ3 ) -30°, the type I
â-turns are found for compounds 7b and 8. The refer-
ence compound 28 only populates this region in confor-
mations above 5 kcal/mol. All conformations of 7a and
the rest of the conformations of 7b and 8 are found in

4528 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
J ohannesson et al.
Sch em e 4a
a
Reagents: (a) (i) His(Trt)-Pro-Phe-Wang resin, HBTU, NMM, DMF, (ii) piperidine, DMF; (b) (i) Fmoc-Tyr(tBu), HBTU, NMM, DMF,
(ii) piperidine, DMF; (c) (i) Fmoc-Cys(Trt), HBTU, NMM, DMF, (ii) piperidine, DMF; (d) (i) Fmoc-Arg(Pmc), HBTU, NMM, DMF, (ii)
piperidine, DMF; (e) (i) Fmoc-Asp(OtBu), HBTU, NMM, DMF, (ii) piperidine, DMF; (f) TFA/H2O; (g) (i) Phe-Wang resin, HBTU, NMM,
DMF, (ii) piperidine, DMF; (h) (i) Fmoc-His(Trt), HBTU, NMM, DMF, (ii) piperidine, DMF; (j) (i) Fmoc-Val, HBTU, NMM, DMF, (ii)
piperidine, DMF; (k) (i) Fmoc-Hcy(Trt), HBTU, NMM, DMF, (ii) piperidine, DMF. Hcy is used as the abbreviation for homocysteine.
the upper left corner of Figure 2b. In the Φ4,Ψ2 plot all
the model compounds of the bicyclic scaffolds seem to
adopt Φ4 values around -80° and Ψ2-values around
150°. As depicted in Figure 2b,c, the linear peptide 28
adopts different backbone torsion angles than 7a , 7b,
and 8. It also seems that the conformational space
available to each of the bicyclic compounds is reduced
as compared to the linear peptide 28.
Finally, we monitored the effect of bicyclization on the
overall geometry of the tripeptidic moieties by analyzing
the torsion angles X1 ) (N2-CR2-CR3-Câ3), X2 ) (Câ3-
CR3-CR4-C(4)O), and X3 ) (N2-CR2-CR4-C(4)O) (Fig-
ure 1). X1, X2, and X3 describe the important directions
of the incoming backbone, the side chain of R2, and the
outgoing backbone with respect to each other. When
these torsion angles were used to analyze the confor-
mational space available to the model compounds, a
slightly different picture emerges (see Figure 3a).
Conformations close to each other in this 3D plot should
have similar geometries with respect to X1, X2, and X3,
but not necessarily with respect to the backbone torsion
angles. As illustrated in Figure 3a, the model compound
of scaffold 7a is located in two different conformational
regions and therefore seems to have the most well-

Angiotensin II Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4529
Ta ble 1. 1H NMR Chemical Shifts for Ang II Analogues 24-26 (DMSO-d6 Solution, 400 MHz)
compd (temp)
residue
NH
H-R
H-â
H-â′
other
24a (35 °C)
Asp
Arg
Cys
Tyr
Aop
His
Pro
Phe
Asp
Arg
Cys
Tyr
Aop
His
Pro
Phe
Asp
Arg
Val
Tyr
Cys
His
Aop
Phe
Asp
Arg
Hcy
Tyr
Aop
His
Pro
Phe
4.10
4.35
4.21
4.87
4.23
4.79
4.38
4.44
4.12
4.53
4.77
4.27
4.20
4.86
4.40
4.43
4.11
4.34
4.13
4.47
4.18
5.06
4.31
4.40
4.11
4.35
4.43
4.52
4.20
4.78
4.39
4.44
2.80
1.60
2.90
3.10
2.14
3.02
2.01
3.03
2.80
1.64
3.91
3.04
2.20
3.11
1.98
3.08
2.79
1.62
1.93
2.80
2.88
3.19
2.12
3.00
2.79
1.65
2.22
2.93
2.06
3.05
2.05
3.03
2.67
1.48
2.84
2.60
1.63
2.90
1.80
2.94
2.68
1.57
3.00
2.87
1.32
3.01
1.71
2.92
2.63
1.49
8.51
8.11
9.03
7.60 (NHꢀ), 3.07 (Hδ, Hδ′), 1.48 (Hγ, Hγ′)
6.99 (2H, ortho), 6.59 (2H, meta)
4.64 (Hδ), 2.24, 1.93 (Hγ, Hγ′)
8.92 (H2), 7.38 (H4)
3.56, 3.45 (Hδ, Hδ′), 1.80 (Hγ, Hγ′)
7.27-7.16 (5H, Ar)
8.28
8.19
24b (35 °C)
25 (25 °C)
26 (25 °C)
8.55
7.64
8.84
7.48 (NHꢀ), 3.06 (Hδ, Hδ′), 1.51 (Hγ, Hγ′)
7.04 (2H, ortho), 6.69 (2H, meta)
5.34 (Hδ), 1.98, 1.89 (Hγ, Hγ′)
8.87 (H2), 7.38 (H4)
3.49 (Hδ, Hδ′), 1.79 (Hγ, Hγ′)
7.27-7.16 (5H, Ar)
7.29
8.02
8.57
7.78
8.00
8.12
9.15
7.66 (NHꢀ), 3.07 (Hδ, Hδ′), 1.50, 1.45 (Hγ, Hγ′)
0.74, 0.75 (Hγ)
7.00 (2H, ortho), 6.61 (2H, meta)
2.64
2.88
2.86
1.75
2.88
2.66
1.49
1.81
2.93
1.89
2.93
1.81
2.94
8.90 (H2), 7.28 (H4)
4.60 (Hδ), 2.26, 1.96 (Hγ, Hγ′)
7.27-7.16 (5H, Ar)
8.18
8.56
7.98
8.49
7.65 (NHꢀ), 3.07 (Hδ, Hδ′), 1.49 (Hγ, Hγ′)
2.67 (Hγ, Hγ′)
7.04 (2H, ortho), 6.66 (2H, meta)
5.52 (Hδ), 2.00, 1.82 (Hγ, Hγ′)
8.91 (H2), 7.42 (H4)
3.56, 3.48 (Hδ, Hδ′), 2.81 (Hγ, Hγ′)
7.27-7.16 (5H, Ar)
8.16
8.28
Ta ble 2. J NH-CR Coupling Constants for Ang II Analogues 24-26 (DMSO-d6 Solution, 400 MHz)a
compd
(temp)
compd
(temp)
compd
(temp)
compd
(temp)
residue
J NH-CR
residue
J NH-CR
residue
J NH-CR
residue
J NH-CR
24a
(35 °C)
Asp
Arg
Cys
Tyr
Aop
His
Pro
Phe
24b
(35 °C)
Asp
Arg
Cys
Tyr
Aop
His
Pro
Phe
25
(25 °C)
Asp
Arg
Val
Tyr
Cys
His
Aop
Phe
26
(25 °C)
Asp
Arg
Hcy
Tyr
Aop
His
Pro
Phe
7.7
7.2
10.3
7.5
6.3
4.6
7.9
8.9
8.0
6.9
10.2
7.9
7.4
7.3
8.0
7.7
7.9
7.7
8.1
7.8
8.0
a
J values are given in hertz (Hz).
Ta ble 3. NMR Temperature Coefficients of NH Chemical
Ta ble 4. In Vitro Receptor Binding Affinities
Shifts for Compounds 24-26 (DMSO-d6 Solution, 400 MHz)a
binding on rat AT1
binding on AT1
receptors expressed by receptors in rat liver
residue residue residue residue residue residue residue
compd
2
3
4
5
6
7
8
compd
CHO cells IC50 (nM)
membranes Ki (nM)
24a
24b
25
2.8
3.7
2.7
2.6
5.6
2.7
4.3
4.3
4.0
3.7
3.7
1.7
b
b
7.0
b
4.4
0.3
4.0
5.4
c
c
b
c
4.8
5.7
4.7
4.0
Ang II
1.5
0.2
40
0.53
0.19
15
c[Hcy3,5]Ang II
DuP 753
24a
24b
25
26
26
>10 000
>10 000
>10 000
800
a
b
∆δ/∆T (ppb/K). No NH (part of the bicyclic ring system). c No
NH (proline).
750
defined geometry of the scaffolds. The model compound
7b can be found in three different regions, while 8 and
especially 28 seem to be more conformationally diverse.
There is one region, indicated with a circle, that is
common to 7a , 7b, and 8 and close also to the reference
compound 28. When the lowest-energy conformation
found in the circle for each of these compounds is
superimposed, as shown in Figure 3b, the overall
topography of these conformations is similar. This
analysis based on the X1-X3 torsion angles is especially
useful for the identification of the scaffold which best
mimics the topographic conformational profile of a
putative target tripeptide moiety.
Discu ssion
We have demonstrated previously24 that an Ang II
derivative with an aspartic acid-derived aldehyde build-
ing block (n ) 1) in the 5-position and cysteine residues
in both positions 3 and 7 afforded regioselective C-
terminal-directed bicyclization (Scheme 5). The ready
formation of the 5-membered ring in favor of 4-, 7-, or
8-membered rings was assumed to provide the driving
force. The one-carbon extension (n ) 2) also gives the
option for bicyclization toward the C-terminal end, but
by formation of a 6-membered ring. However, cyclization

4530 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
J ohannesson et al.
Ch a r t 3
F igu r e 1. Parameters used to characterize the scaffolds. The
conformation of model compound 7b shown in the figure adopts
a type I â-turn. The relative steric energy of this conformation
is ∆E ) 1.0 kcal/mol.
in the opposite direction, toward the N-terminal end, is
now strongly favored, and formation of the 5-membered
ring as opposed to the 6-membered ring seems to govern
the regioselection. Thus, the regioselectivity of the
bicyclization can be altered, simply by varying the chain
length of the incorporated aldehyde precursor (Scheme
5). While the C-terminal-directed bicyclization (n ) 1)
for the production of rigid tripeptide mimetics proceeds
with very high stereoselectivity, both epimers are
formed in some cases with N-terminal-directed bicy-
clizations.
Marshall’s and Baldwin’s groups have reported two
complementary bicyclization methodologies for the prepa-
ration of bicyclic dipeptide scaffolds related to ours, but
with the inclusion of oxygen in the larger ring. Mar-
shall’s procedure46-48 relied on an anodic oxidation of a
proline residue as a key step. Baldwin49 used (S)-but-
3-enylglycine as aldehyde precursor, reacting with an
L-serine hydroxyl group, to form the oxazabicycloalkane
system. An elegant example of an aldehyde side chain
to N-terminal cyclization, to afford a thiazolidine fused
to a large ring, consisting of 10 amino acid residues, has
been reported by Tam’s group.50
In the previous study, we demonstrated that the
bicyclic tripeptide scaffolds 5 and 6 preferentially induce
â-turn geometries of the nonclassical type.24 In the
present study we report that scaffolds 7a , 7b, and 8 also
can adopt â-turns, of which some correspond to the type
I â-turn. When the distribution of backbone torsion
F igu r e 2. (a) Scatter plot of CR1-CR4 and CR2-CR5 distances
for all conformations below 5 kcal/mol of the lowest-energy
conformation for model compounds 7a , 7b, 8, 27, and 28.
Conformations to the left of the red line have CR1-CR4
distances below 7 Å, and conformations below the blue line
have CR2-CR5 distances below 7 Å. (b) Scatter plot of backbone
torsion angles Φ3 and Ψ3 for all conformations below 5 kcal/
mol of the lowest-energy conformation for model compounds
7a , 7b, 8, 27, and 28. (c) Scatter plot of backbone torsion angles
Φ4 and Ψ2 for all conformations below 5 kcal/mol of the lowest-
energy conformation for 7a , 7b, 8, 27, and 28.
angles are analyzed, a different conformational distri-
bution, as well as a restriction in conformational space,
can be seen for the bicyclic model compounds 7a , 7b,
and 8 as compared to the linear peptide 28.

Angiotensin II Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4531
F igu r e 3. (a) Stereo 3D plot of torsion angles X1 ) (N2-CR2-CR3-Câ3), X2 ) (Câ3-CR3-CR4-C(4)O), and X3 ) (N2-CR2-CR4-
C(4)O) for all conformations below 5 kcal/mol of the lowest-energy conformation for model compounds 7a , 7b, 8, 27, and 28. (b)
Stereo image of the rms best fit of the lowest-energy conformations found within the circle indicated in the 3D plot in part a
above for each of the model compounds 7a , 7b, 8, and 28. Color scheme: 7a (blue) ∆E ) 0 kcal/mol, 7b (red) ∆E ) 1.2 kcal/mol,
8 (green) ∆E ) 1.2 kcal/mol, and 28 (black) ∆E ) 2.1 kcal/mol. N2, CR2, CR3, Câ3, CR4, and C(4)O were included in the fitting
procedure.
Sch em e 5a
a
The regioselectivity of the bicyclization can be altered, simply by varying the chain length of the incorporated aldehyde precursor.
It is generally accepted that in Ang II, Val3, Ile5, and
Pro7 have conformational stabilizing roles.51 Marshall
et al. have demonstrated that bicylization constraints
in the 3-5 and 5-7 regions delivered the high-affinity
Ang II analogues 3 and 4. We were encouraged therefore
to incorporate the new aldehyde precursor in both
positions 5 and 7, to enforce constraint into these two
regions. The receptor binding studies of 24-26 showed
that Ang II analogue 26, where the bicyclization had
been executed in the 3-5 region, exhibited affinity,
although weak (Ki ) 750 nM), to the AT1 receptor. The
most potent of the Ang II analogues synthesized by
Marshall et al., the bicyclic disulfide 3 (IC50 ) 1.3 nM),11
differs from the most potent in our series (i.e., 26) with
respect to the position of the sulfur atom and the
stereochemistry at the ring junction of the bicyclic
system, and also with respect to position 1, where a Sar
residue is present in 3. Such a Sar residue is known to
enhance the receptor binding affinity of Ang II ana-
logues 3-10 times14,52 and can therefore account for part
of the difference in receptor binding affinity between 3
and 26.
The major reason for the difference in binding affinity
between these two octapeptides is more likely due to
conformational effects. Conformational studies with
NMR have previously supported the presence of mul-
tiple conformers for Ang II and Ang II analogues in
solution.16,53-56 NMR data (DMSO-d6) and independent
energy calculations have been presented for Ang II
analogue 3.16 These NMR data enable a qualitative

4532 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
J ohannesson et al.
F igu r e 4. Stereo image of the rms best fit of the lowest energy conformations of 8 (green) and 27 (black). N2, CR2, CR3, Câ3, CR4,
and C(4)O were included in the fitting procedure. Mean average distance between fitted atoms ) 0.25 Å.
comparison between the conformational profiles of the
octapeptides 3 and 26. This comparison shows that the
NH temperature coefficients show essentially the same
trend for 26 as compared to 3, namely that the lowest
coefficients are observed for the Arg- and Tyr-NH
protons (in 3:16 2.8 and 2.7, in 26: 2.6 and 1.7). The
lower value for the Tyr-NH proton in Ang II analogue
26 could indicate an involvement in an intramolecular
hydrogen bond, whereas this is less pronounced in 3.
The NH to CR coupling constants are smaller for 3 than
for 26, except for Tyr where a higher value of 9.3 Hz is
observed for octapeptide 3 (cf. J ) 7.3 Hz for 26). Taken
together, the two octapeptides have similar conforma-
tional profiles based on their NMR data.
To compare the conformational preferences of these
octapeptides further, we analyzed the Φ and Ψ plots
(Figure 2b,c) and the X1-X3 plot (Figure 3a) obtained
for the corresponding tripeptidic model compounds 8
and 27. In this analysis we assumed that the confor-
mational preferences of the bicyclic scaffolds would not
change significantly when they are inserted into Ang
II. Thus, if the overall geometry of these scaffolds is
similar, octapeptides 3 and 26 should (at least based
on conformational reasoning) be able to present the
pharmacophore groups in the same 3D arrangement.
In fact, conformations with similar torsion angles can
be found for 27 and all the other bicyclic model com-
pounds. The overall similarities in topography of model
compounds 8 and 27 are shown in Figure 3a and are
illustrated further in Figure 4, in which the lowest-
energy conformation of 8 and 27 is superimposed. In
summary, our conformational analysis of the model
compounds does not allow for rationalization of the
difference in affinity between Ang II analogues 3 and
26.
aspartic acid-derived precursor to induce cyclization
toward the C-terminal end of the peptide. Thus, the
regioselectivity of the bicyclization can be reversed,
simply through variation of the chain length of the
incorporated aldehyde precursor. Insertion of these new
bicyclic tripeptide scaffolds into the 3-5- and 5-7-
positions of Ang II delivered one analogue (26) with an
AT1 receptor affinity of Ki ) 750 nM. This Ang II
analogue is structurally very similar to the highly potent
bicyclic mercaptoproline derivative 3 (IC50 ) 1.3 nM),
suggesting that minor structural alterations have a
large impact on the affinity.
Although only a few examples of the application of
the presented bicyclization concept are given herein, we
believe that the procedure should be amenable to
modifications to allow for the introduction of side chains
other than Tyr and His in the turn region. The proce-
dure should be applicable also to the elaborations of
other target peptides and should hopefully serve as a
valuable research tool for the medicinal chemist.
Exp er im en ta l Section
Ch em istr y. Gen er a l Com m en ts. 1H and 13C NMR spectra
were recorded on a J EOL J NM-EX270 at 270 (67.8) MHz, on
a J EOL J NM-EX400 at 400 (100.5) MHz, or on a Varian Unity
400 spectrometer at 400 (100.6) MHz. Spectra were recorded
at ambient temperature unless otherwise noted. Chemical
shifts are reported as δ values (ppm), referenced to Me4Si. IR
spectra were recorded on a Perkin-Elmer model 1605 FT-IR
instrument and are reported as νmax (cm-1). Optical rotations
were measured at ambient temperature on a Perkin-Elmer
model 241 polarimeter. Melting points (uncorrected) were
determined in open glass capillaries in
a melting point
microscope. Elemental analyses were performed by Mikro
Kemi AB, Uppsala, Sweden. High-resolution mass spectros-
copy (HRMS) was performed by Dr. S. Gohil, Department of
Chemistry, SLU, Uppsala, Sweden. Flash column chromatog-
raphy was performed using Merck silica gel 60 (40-63 µm) or
Riedel-de Hae¨n silica gel S (32-63 µm). Thin-layer chroma-
tography (TLC) was performed using aluminum sheets pre-
coated with silica gel 60 F254 (0.2 mm, E. Merck, or 0.25 mm,
Macherey-Nagel). Chromatographic spots were visualized by
UV and/or spraying with an ethanolic solution of ninhydrin
(2%) followed by heating. Mass spectroscopy was carried out
on an Applied Biosystems (Uppsala, Sweden) BIOION 20
plasma desorption mass spectrometer. Amino acid analyses
and peptide content determinations were performed by Dr. M.
Sundquist, Department of Biochemistry, Biomedical Centre,
Uppsala, Sweden, on 24-h hydrolyzates with an LKB 4151
alpha plus analyzer, using ninhydrin detection.
Subtle geometrical variations in the scaffolds appar-
ently affect the affinity, which in turn is largely deter-
mined by the relative spatial orientation of side-chain
pharmacophores. Thus, access to an armory of simple,
complementary synthetic procedures which provides a
diversity of scaffolds, including classical secondary
structure mimetics, and that allows the full conforma-
tional space to be covered is highly desirable in the wait
for 3D structural data of peptide/G-protein-coupled
receptors.
Con clu sion
We have developed an experimentally simple proce-
dure on solid phase for the construction of new tripep-
tidic 5,9- and 5,10-fused thiazabicycloalkane tripeptide
mimetics. This N-terminal-directed bicyclization, relying
on masked aldehyde precursors derived from L-glutamic
acid as key building blocks, provides a complement to
the previously reported related bicyclization using an
Ma ter ia ls. SPPS resins and amino acid derivatives were
obtained from Bachem (Bubendorf, Switzerland), Calbiochem-
Novabiochem (La¨ufelfingen, Switzerland), or Alexis Corp.
(La¨ufelfingen, Switzerland). DMF (peptide synthesis grade)
was obtained from PerSeptive Biosystems (Hamburg, Ger-
many) and was used without further purification. 2-(1H-
Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-

Angiotensin II Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4533
phate (HBTU) and 1-hydroxybenzotriazole (HOBt) were pur-
chased from Richelieu Biotechnologies (St-Hyacinthe, Qc,
Canada). Monoiodinated [125I]Ang II (2000 Ci/mmol) was
purchased from Euro-Diagnostica AB, Malmo¨, Sweden, and
was prepared by the chloramine-T method.
(C(CH3)3, 31.7, 32.8 (4), 59.2, 59.6 (2), 78.8, 79.7, 80.0 (C(CH3)3),
80.6, 81.2 (5), 152.7 (CO Boc), 171.0 (CO carboxylic acid); IR
(neat) 3454, 1741, 1703. Anal. (C14H25NO5) C, H, N.
N-(9-F lu or en ylm et h yloxyca r b on yl)-5-h yd r oxy-L-p r o-
lin e (12). The proline derivative 11 (0.42 g, 1.5 mmol) was
dissolved in CH2Cl2 under N2 atmosphere at room tempera-
ture. TMS-I (0.70 mL, 4.9 mmol) was added and the mixture
was stirred at room temperature. After 50 min the reaction
was quenched with MeOH (3.6 mL, 89 mmol) and stirred for
another 20 min. The reaction mixture was evaporated and the
yellow residue was redissolved in a mixture of 10% aqueous
Na2CO3 (30 mL) and dioxane (15 mL) and cooled to 0 °C. Fmoc-
Cl (0.57 g, 2.2 mmol) was dissolved in dioxane (15 mL) and
added dropwise, whereafter the reaction was allowed to reach
room temperature. The pH was kept around 10-11. After the
mixture was stirred at room temperature for 96 h, 10%
aqueous citric acid was added to pH 8, and the reaction
mixture was washed with ether until TLC showed the water
phase to be free from impurities having Rf values higher than
those of the product (4 × 100 mL). Ether (200 mL) was added
to the water phase, which was then acidified to pH 2-3 with
10% aqueous citric acid, under vigorous stirring. The phases
were separated and the water phase was further extracted
with ether (3 × 150 mL). The combined organic layers were
washed with water (2 × 100 mL), dried (MgSO4), and concen-
trated to yield the building block 12 (0.21 g, 41%) as a white
foam. NMR spectra show the existence of each diastereoisomer
as a pair of rotamers: TLC Rf ) 0.20 and 0.30 for the two
Solid -P h a se P ep tid e Syn th esis (SP P S). The peptides
were synthesized on a 60-80-µmol scale with a Symphony
instrument (Protein Technologies Inc., Tucson, AZ) using
Fmoc/tert-butyl protection. The starting polymer was Fmoc-
Phe-Wang resin (0.6 mmol/g), and for the Fmoc amino acids
the side chain protecting groups were as follows: Asp(OtBu),
Arg(Pmc), Tyr(tBu), His(Trt), Cys(Trt), and Hcy(Trt). Removal
of the Fmoc group was achieved by reaction with 20% piperi-
dine in DMF for 5 + 10 min. Unless otherwise stated, coupling
of the amino acids (125 µmol) was done in DMF (2.5 mL) using
HBTU (125 µmol) in the presence of NMM (0.5 mmol). Single
couplings (60 min) were used for Fmoc-Hcy(Trt) and for
compound 20, double couplings (2 × 30 min) for the other
amino acids. After the introduction of each amino acid,
remaining amino groups were capped by addition of 20% acetic
anhydride in DMF (1.25 mL) to the coupling mixture and
allowing the reaction to proceed for 5 min. No capping was
performed in cases where 5-hydroxyproline was present in the
resin-bound peptide. After completion of the synthesis, the
Fmoc group was removed and the partially protected peptide
resin was washed with several portions of DMF and CH2Cl2
and dried in a stream of nitrogen and in vacuo. Yields for the
purified Ang II analogues were corrected for peptide content.
diastereoisomers, respectively (CH2Cl2/MeOH 9:1); [R]D
)
L-2-(ter t-Bu t oxyca r b on yla m in o)-4-(N-m et h oxy-N-m e-
th ylca r ba m oyl)bu ta n oic Acid ter t-Bu tyl Ester (10).57 Boc-
Glu-OtBu (9) (8.57 g, 28.3 mmol), NEt3 (4.3 mL, 31 mmol), and
PyBOP (16.2 g, 31.1 mmol) were dissolved in CH2Cl2 and
stirred at room temperature for 10 min. N,O-Dimethylhy-
droxylamine hydrochloride (3.18 g, 32.6 mmol) and a second
portion of NEt3 (4.5 mL, 33 mmol) were added and the mixture
was stirred at room temperature for 48 h. The reaction mixture
was washed with 10% aqueous citric acid (2 × 90 mL),
saturated aqueous NaHCO3 (2 × 150 mL), and brine (1 × 100
mL). The organic phase was dried (MgSO4) and evaporated.
The residue was purified by flash chromatography (gradient
system: pentane/EtOAc 7:3 to pentane/EtOAc 11:9) to give 10
as white crystals (9.06 g, 92%): mp 58-60 °C; TLC Rf ) 0.45
-43.1° (c ) 1.0, 99% EtOH); 1H NMR (CDCl3, 20 °C) δ 1.70-
2.61 (m, 4H, 3 and 4), 4.08-4.78 (m, 5H, OH, 2, CH Fmoc and
CH2 Fmoc), 5.04 (br d), 5.27 (br s) and 5.64 (br d) (1H together,
5), 7.23-7.82 (m, 8H, CH Ar Fmoc); 13C NMR (CDCl3, 20 °C)
δ 26.5, 26.9, 28.0, 30.2, 30.9, 31.6, 32.0, 33.2 (3 and 4), 46.8,
47.0 (CH Fmoc), 58.6, 58.8 (2), 66.9, 67.0, 67.9 (CH2 Fmoc),
81.6, 82.2, 82.3, 82.9 (5), 119.9, 120.0, 120.1, 124.5, 124.7,
124.8, 124.9, 127.0, 127.3, 127.7, 127.8, 127.9 (CH Ar Fmoc),
140.0, 141.11, 141.14, 141.2, 143.30, 143.35, 143.5, 143.6 (C
ipso Ar Fmoc), 154.2, 154.8 (CO Fmoc), 176.0, 176.7 (CO
carboxylic acid); IR (KBr) 3430, 1704. Anal. (C20H19NO5‚
1.25H2O) C, N; H: calcd, 5.76; found, 5.1. HRMS (FAB) calcd
for C20H19NO5 (M + H+): 354.1342. Found: 354.1351.
L-3-(Ben zyloxyca r bon yl)-4-(3,3-d im eth oxyp r op yl)-1,3-
oxa zolid in -5-on e (17).32 L-3-(3-(Benzyloxycarbonyl)-5-oxo-1,3-
oxazolidin-4-yl)propanoic acid (14) (26.4 g, 90.0 mmol) was
converted to L-3-(3-(benzyloxycarbonyl)-5-oxo-1,3-oxazolidin-
4-yl)propanal (16)59-61 as described in the literature.31 Crude
16 (18.9 g) and p-toluenesulfonic acid (monohydrate, 98.5%)
(0.65 g, 3.4 mmol) were dissolved in MeOH (1 L). After stirring
at room temperature for 2 h, most of the solvent was
evaporated and the residue was partitioned between EtOAc
(300 mL) and saturated aqueous NaHCO3 (100 mL). The
organic phase was washed with brine (100 mL), dried (MgSO4),
and concentrated. Purification by flash chromatography (gra-
dient system: pentane/EtOAc 4:1 to pentane/EtOAc 2:1) gave
the product 17 as a colorless oil (14.5 g, 50% over three steps,
from compound 14): TLC Rf ) 0.65 (pentane/EtOAc 3:2); [R]D
1
(pentane/EtOAc 1:1); [R]D ) -18.9° (c ) 1.0, 99% EtOH); H
NMR (CDCl3) δ 1.43 (s, 9H, C(CH3)3), 1.46 (s, 9H, C(CH3)3),
1.83-1.98 (m, 1H, CH2), 2.06-2.22 (m, 1H, CH2), 2.39-2.62
(m, 2H, CH2), 3.17 (s, 3H, NCH3), 3.67 (s, 3H, OCH3), 4.19 (m,
1H, 2), 5.18 (br d, J ) 7.6 Hz, 1H, NH); 13C NMR (CDCl3) δ
27.4 (CH2), 27.8 (C(CH3)3), 27.9 (CH2), 28.2 (C(CH3)3), 32.1
(NCH3), 53.6 (2), 61.1 (OCH3), 79.4, 81.7 (C(CH3)3), 155.4 (CO
Boc), 171.4, 173.4 (CO ester and CO carbamoyl); IR (KBr) 3267,
1732, 1708, 1639. Anal. (C16H30N2O6) C, H, N.
N-(ter t-Bu toxyca r bon yl)-5-h yd r oxy-L-p r olin e ter t-Bu -
tyl Ester (11).25,58 Amide 10 (5.00 g, 14.4 mmol) was dissolved
in dry THF, under N2 atmosphere, and cooled to -78 °C.
DIBALH (1 M in hexane) (21.7 mL, 21.7 mmol) was added
dropwise during 15 min, whereafter the reaction mixture was
stirred at -78 °C for 1.5 h. The reaction mixture was
partitioned between ether (135 mL) and 10% aqueous citric
acid (75 mL). The aqueous phase was further extracted with
ether (100 mL). The combined organic layers were washed with
10% aqueous citric acid (1 × 65 mL) and brine (1 × 65 mL),
dried (MgSO4), and evaporated. Purification by flash chroma-
tography (gradient system: pentane/EtOAc 9:1 to pentane/
EtOAc 3:2) afforded the pure product 11 (3.70 g, 89%), colorless
oil, as a pair of diastereoisomers (approximate ratio 1:1). Some
peaks in the NMR spectra are duplicated, due to hindered
rotation of the carbamate function:28,29 TLC Rf ) 0.45 (pentane/
EtOAc 7:3); [R]D ) -62.8° (c ) 1.0, 99% EtOH); 1H NMR
(DMSO-d6, 90 °C) δ 1.34-1.54 (m, 18H, (C(CH3)3), 1.60-2.45
(m, 4H, 3 and 4), 3.98-4.10 (m, 1H, 2), 4.93 and 5.27 (2 br s,
1H together, exchangeable with D2O, OH), 5.33-5.44 (m, 1H,
5); 13C NMR (DMSO-d6, 90 °C) δ 27.0 (3), 27.4, 27.5, 27.8
1
) +82.2° (c ) 1.0, CHCl3); H NMR (CDCl3) δ 1.59-1.78 (m,
2H, CH2), 1.86-2.24 (m, 2H, CH2), 3.27 (s, 6H, CH(OCH3)2),
4.28-4.38 (br m, 2H, 3 and 4), 5.12-5.26 (m, 3H, OCHaN and
OCH2Ar), 5.52 (br s, 1H, OCHbN), 7.35-7.39 (m, 5H, CH Ar);
13C NMR (CDCl3) δ 25.4, 27.0 (CH2CH2), 52.5, 52.6 (CH-
(OCH3)2), 54.2 (4), 67.6 (OCH2Ar), 77.6 (OCH2N), 103.5 (3),
128.0, 128.3, 128.4 (CH Ar), 135.2 (ipso Ar), 152.6 (CO Cbz),
171.9 (5); IR (neat) 1803, 1719. Anal. (C16H21NO6) C, H, N.
L-2-(Ben zyloxyca r bon yla m in o)-5,5-d im eth oxyp en ta n o-
ic Acid Meth yl Ester (18).32 Dimethyl acetal 17 (12.0 g, 37.2
mmol) was dissolved in MeOH (500 mL) under N2 atmosphere
and cooled to -12 °C. Sodium methoxide (2.00 g, 37.0 mmol)
was suspended in MeOH (45 mL) and added dropwise during
1 h. After stirring for 2 h, aqueous citric acid was added to pH
7-8, and half the amount of solvent was evaporated. The
residue was poured into EtOAc (500 mL) and washed with 10%

4534 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
J ohannesson et al.
aqueous NaCl (2 × 500 mL). The organic layer was dried
(MgSO4) and concentrated and the residue was purified by
flash chromatography (gradient system: pentane/EtOAc 5:1
to pentane/EtOAc 3:2) to yield 18 (10.7 g, 89%) as a colorless
oil: TLC Rf ) 0.60 (pentane/EtOAc 3:2); [R]D ) +7.5° (c ) 1.0,
CHCl3); 1H NMR (CDCl3) δ 1.56-1.80 (m, 3H, 3a, 4), 1.85-
1.98 (m, 1H, 3b), 3.29 (s, 3H, CHOCH3), 3.30 (s, 3H, CHOCH3),
3.74 (s, 3H, COOCH3), 4.34 (app t, 1H, 5), 4.39 (app dd, 1H,
2), 5.11 (s, 2H, OCH2Ar), 5.38 (br d, J ) 7.8 Hz, 1H, NH), 7.29-
7.39 (m, 5H, CH Ar); 13C NMR (CDCl3) δ 27.3, 28.2 (3, 4), 52.2
(COOCH3), 52.8 (CH(OCH3)2), 53.4 (2), 66.8 (OCH2Ar), 103.7
(5), 127.9, 128.0, 128.4 (CH Ar), 136.1 (ipso Ar), 155.8 (CO Cbz),
172.6 (1); IR (neat) 3330, 1721. Anal. (C16H23NO6) C, H, N.
monotritylated. Therefore, the product was dissolved in TFA
(0.75 mL) and a 5% solution of triethylsilane (0.75 mL) was
added to produce a nearly colorless solution. Precipitation and
washing with ether as described above furnished 44.3 mg
(67.7%) of the crude bicyclic Ang II analogue 23. The peptide
(39.4 mg) was dissolved in 0.1% aqueous TFA and purified in
three runs by RP-HPLC on a Vydac 10-µm C18 column (1.0 ×
25 cm) with an 80-min gradient of 10-50% MeCN in 0.1%
aqueous TFA at a flow rate of 3 mL/min. The separation was
monitored at 230 nm and by PDMS. Two compounds of the
expected mass were isolated. 23a : 2.9 mg (5.1%); PDMS (MW
1038.0) 1038.7 (M + H+). 23b: 2.2 mg (3.8%); PDMS 1039.0
(M + H+).
L-2-Am in o-5,5-d im eth oxyp en ta n oic Acid Meth yl Ester
(19).32,62 Compound 18 (10.5 g, 32.2 mmol) and 10% Pd/C (1.74
g, 1.64 mmol) were mixed in absolute ethanol and stirred
under H2 (1 atm) at room temperature for 2 h. The mixture
was filtered through Celite and concentrated. The residue was
purified by flash chromatography (gradient system: CH2Cl2
to CH2Cl2/MeOH 24:1) to give the product 19 (5.08 g, 83%) as
a colorless oil: TLC Rf ) 0.40 (CH2Cl2/MeOH 9:1); [R]D ) +16°
Meth od 2, u sin g F m oc-L-2-a m in o-5,5-d im eth oxyp en -
ta n oic a cid (20): The peptide was synthesized according to
the same procedure as in method 1, using PyBOP/HOBt/DIEA
for the incorporation of amino acid derivative 20 and for the
tyrosine residue. The partially protected peptide resin, 215 mg
(95.0%), was cleaved as described above to yield 51.7 mg
(79.4%) of crude detritylated product. This material was
dissolved in 0.1% aqueous TFA (12 mL) and purified by two
runs on a Vydac 10-µm C18 column (2.2 × 25 cm) using an
80-min gradient of 0-40% MeCN in 0.1% aqueous TFA at a
flow rate of 4 mL/min. Again, two compounds of the expected
mass were obtained. 23a : 5.7 mg (5.7%); PDMS (MW 1038.0)
1039.0 (M + H+); amino acid analysis Asp, 0.99; Arg, 0.99;
Tyr, 0.99; His, 1.01; Phe, 1.01; Cys, not determined (nd) (66%
peptide); 1H NMR (DMSO-d6, 25 °C, 400 MHz) δ 1.44 (m, 2H,
Hγ-Arg, Hγ′-Arg), 1.49 (m, 1H, Hâ-Arg), 1.59 (m, 1H, Hâ′-Arg),
1.74 (m, 1H, Hâ-Aop), 1.93 (m, 1H, Hγ-Aop), 2.11 (m, 1H, Hâ′-
Aop), 2.22 (m, 1H, Hâ-Cys7), 2.60 (m, 1H, Hâ-Tyr), 2.68 (m,
1H, Hâ-Asp), 2.70 (m, 1H, Hâ′-Cys7), 2.77 (m, 1H, Hâ′-Asp),
2.77 (m, 1H, Hγ′-Aop), 2.90 (m, 2H, Hâ-Cys3, Hâ′-Cys3), 2.90
(m, 1H, Hâ-Phe), 2.95 (m, 1H, Hâ-His), 3.04 (m, 1H, Hâ′-Phe),
3.06 (m, 2H, Hδ-Arg, Hδ′-Arg), 3.07 (m, 1H, Hâ′-His), 3.15 (m,
1H, Hâ′-Tyr), 4.10 (m, 1H, HR-Asp), 4.21 (m, 1H, HR-Cys3),
4.25 (dm, J ) 9.5 Hz, 1H, HR-Aop), 4.34 (m, 1H, HR-Arg), 4.41
(m, 1H, HR-Cys7), 4.43 (m, 1H, HR-Phe), 4.52 (ddd, J ) 8.4,
7.9, 5.6 Hz, 1H, HR-His), 4.63 (dd, J ) 4.8, 1.8 Hz, 1H, Hδ-
Aop), 4.84 (ddd, J ) 10.2, 9.6, 4.7 Hz, 1H, HR-Tyr), 6.57 (m,
2H, meta-Tyr), 7.00 (m, 2H, ortho-Tyr), 7.17-7.26 (m, 5H,
Phe), 7.32 (br s, 1H, H4-His), 7.57 (m, 1H, NHꢀ-Arg), 7.76 (d,
J ) 7.9 Hz, 1H, NH-Cys7), 8.18 (d, J ) 7.1 Hz, 1H, NH-Cys3),
8.38 (d, J ) 7.8 Hz, 1H, NH-Phe), 8.43 (d, J ) 8.4 Hz, 1H,
NH-His), 8.54 (d, J ) 7.6 Hz, 1H, NH-Arg), 8.90 (br s, 1H,
H2-His), 9.09 (d, J ) 10.2 Hz, 1H, NH-Tyr). 23b: 9.0 mg
(10.4%); PDMS 1039.8 (M + H+); amino acid analysis Asp,
1.00; Arg, 1.00; Tyr, 0.99; His, 1.01; Phe, 1.00; Cys, nd (75%
peptide); 1H NMR (DMSO-d6, 25 °C, 400 MHz) δ 1.26 (m, 1H,
Hâ-Aop), 1.48 (m, 1H, Hâ-Arg), 1.48 (m, 2H, Hγ-Arg, Hγ′-Arg),
1.63 (m, 1H, Hâ′-Arg), 1.90 (m, 1H, Hγ-Aop), 1.97 (m, 1H, Hγ′-
Aop), 2.18 (m, 1H, Hâ′-Aop), 2.48 (m, 1H, Hâ-Cys7), 2.65 (m,
1H, Hâ-Asp), 2.72 (m, 1H, Hâ′-Cys7), 2.76 (m, 1H, Hâ′-Asp),
2.90 (m, 1H, Hâ-Tyr), 2.90 (m, 1H, Hâ-Phe), 2.96 (m, 1H, Hâ-
Cys3), 2.96 (m, 1H, Hâ-His), 3.00 (m, 1H, Hâ′-Phe), 3.03 (m,
2H, Hδ-Arg, Hδ′-Arg), 3.06 (m, 1H, Hâ′-Tyr), 3.35 (m, 1H, Hâ′-
His), 3.93 (m, 1H, Hâ′-Cys3), 4.10 (m, 1H, HR-Asp), 4.21 (dd,
J ) 10.2, 8.5 Hz, 1H, HR-Aop), 4.39 (m, 1H, HR-Cys7), 4.41
(m, 1H, HR-Arg), 4.41 (m, 1H, HR-Phe), 4.43 (m, 1H, HR-Tyr),
4.67 (ddd, J ) 10.2, 8.6, 4.5 Hz, 1H, HR-His), 4.81 (ddm, J )
6.6, 4.6 Hz, 1H, HR-Cys3), 5.30 (dd, J ) 5.7, 1.8 Hz, 1H, Hδ-
Aop), 6.69 (m, 2H, meta-Tyr), 7.07 (m, 2H, ortho-Tyr), 7.17-
7.25 (m, 5H, Phe), 7.44 (m, 1H, NH-His), 7.44 (m, 1H, H4-
His), 7.46 (m, 1H, NH-Cys7), 7.65 (m, 1H, NHꢀ-Arg), 8.13 (d,
J ) 7.7 Hz, 1H, NH-Phe), 8.36 (br, 1H, NH-Cys3), 8.59 (d, J )
7.6 Hz, 1H, NH-Arg), 8.85 (br, 1H, H2-His), 8.89 (d, J ) 3.7
Hz, 1H, NH-Tyr).
1
(c ) 1.0, CHCl3); H NMR (CDCl3) δ 1.47-1.86 (m, 6H, 3, 4
and NH2), 3.30 (s, 3H, CHOCH3), 3.31 (s, 3H, CHOCH3), 3.44
(dd, J ) 5.2, 7.4 Hz, 1H, 2), 3.71 (s, 3H, COOCH3), 4.36 (t, J
) 5.3 Hz, 1H, 5); 13C NMR (CDCl3) δ 28.5 (4), 29.7 (3), 51.8
(COOCH3), 52.6, 52.7 (CH(OCH3)2), 54.0 (2), 104.0 (5), 176.2
(1); IR (neat) 3376, 1737. Anal. (C8H17NO4) C, H, N.
L-2-((9-F lu o r e n y lm e t h y lo x y c a r b o n y l)a m in o )-5,5-
d im eth oxyp en ta n oic Acid (20).63 To compound 19 (0.48 g,
2.5 mmol) dissolved in MeOH (30 mL) was added 1 M aqueous
KOH (2.5 mL, 2.5 mmol) and the mixture was stirred at room
temperature for 3.5 h; 10% aqueous citric acid was added to
pH 7-8 and the reaction mixture was concentrated to give a
solid residue. This solid residue was dissolved in a mixture of
10% aqueous Na2CO3 (45 mL) and dioxane (23 mL) and cooled
to 0 °C. Fmoc-Cl (0.97 g, 3.7 mmol) dissolved in dioxane (23
mL) was added dropwise, whereafter the reaction was allowed
to reach room temperature. The pH was kept around 10-11.
Stirring at room temperature was continued for 96 h. The
workup was performed through extensive extractions following
essentially the same procedure as described for compound 12
to afford pure product 20 as a white foam (0.62 g, 62%): TLC
Rf ) 0.45 (CH2Cl2/MeOH 9:1); [R]D ) +8.6° (c ) 1.0, CHCl3);
1H NMR (acetone-d6) δ 1.65-1.81 (m, 3H, 3a and 4), 1.86-
1.97 (m, 1H, 3b), 3.27 (s, 3H, CHOCH3), 3.28 (s, 3H, CHOCH3),
4.22-4.31 (m, 4H, CH Fmoc, 2 and CH2 Fmoc), 4.41 (app t,
1H, 5), 6.85 (br d, J ) 8.0 Hz, 1H, NH), 7.32 (t, J ) 7.6 Hz,
2H, CH Ar Fmoc), 7.41 (t, J ) 7.6 Hz, 2H, CH Ar Fmoc), 7.72
(app t, J ) 6.9 Hz, 2H, CH Ar Fmoc), 7.85 (d, J ) 8.7 Hz, 2H,
CH Ar Fmoc); 13C NMR (acetone-d6) δ 27.5, 29.5 (3, 4), 47.9
(CH Fmoc), 52.8, 53.0 (CH(OCH3)2), 54.4 (2), 67.1 (CH2 Fmoc),
104.7 (5), 120.7, 126.0, 126.1, 127.8, 128.4 (CH Ar Fmoc), 141.9,
144.8, 145.0 (ipso Fmoc), 157.0 (CO Fmoc), 173.9 (1); IR (KBr)
3324, 1720. Anal. (C22H25NO6‚H2O) C, H, N.
An g II An a logu es 23a a n d 23b. Meth od 1, u sin g F m oc-
5-h yd r oxy-L-p r olin e (12): Fmoc-Phe-Wang resin (105 mg,
63 µmol) was reacted with the appropriate amino acids as
described above (general procedure). However, the hydrox-
yproline derivative 12 (125 µmol) was coupled for 19.5 h using
PyBOP (125 µmol) in the presence of HOBt (125 µmol) and
diisopropylethylamine (DIEA) (313 µmol). The next residue,
Fmoc-Tyr(OtBu)-OH, was coupled for 4 h in the same way but
using 5 equiv of amino acid, PyBOP, HOBt, and 10 equiv of
DIEA. The yield of the partially protected peptide resin was
212 mg (95.6% according to weight increase). The resin was
treated with 95% aqueous TFA (2.5 mL) for 2 h and the
mixture was then filtered through a small plug of glass wool
in a Pasteur pipet. After washing with TFA (3 × 0.3 mL), the
combined filtrates were evaporated in a stream of dry nitrogen
to ca. 1.5 mL and the product was precipitated by the addition
of cold, anhydrous ether (12 mL). The precipitate was collected
by centrifugation, washed with ether (4 × 7 mL), and dried.
Plasma desorption mass spectroscopy (PDMS) analysis re-
vealed that a substantial part of the crude material was still
An g II An a logu es 24a a n d 24b. Automated synthesis on
an 80-µmol scale according to the general procedure and using
20 for incorporation of the Aop residue provided 245 mg
(97.4%) of partially protected resin which was cleaved and
deprotected with 95% aqueous TFA (2.5 mL) for 1.5 h. The
resin was filtered off and washed with 5% triethylsilane in
TFA (3 × 0.3 mL). The peptide was precipitated with cold ether

Angiotensin II Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4535
(10 mL), collected by centrifugation, washed with ether (4 ×
7 mL), and dried to yield 71.2 mg (90.5%). The crude material
was divided in four aliquots, each dissolved in 0.1% aqueous
TFA, containing 15% MeCN (5 mL) and chromatographed on
a Vydac 10-µm C18 column (1 × 25 cm) using a 60-min
gradient of 15-45% MeCN in 0.1% aqueous TFA at a flow rate
of 3 mL/min. The fractions corresponding to the two major
peaks both contained material of the expected mass. 24a : 19.2
mg (17.0%); PDMS (MW 1032.1); 1033.5 (M + H+); amino acid
analysis Asp, 1.01; Arg, 0.98; Tyr, 0.99; His, 0.99; Pro, 1.01;
Phe, 1.02; Cys, nd (73% peptide). 24b: 28.1 mg (26.6%); PDMS
1033.7 (M + H+); amino acid analysis Asp, 1.01; Arg, 1.02;
Tyr, 0.97; His, 0.98; Pro, 1.02; Phe, 1.00; Cys, nd (78% peptide).
Con for m a tion a l En er gy Ca lcu la tion s. The calculations
of 7a , 7b, 8, 27, and 28 were performed using the Amber* all-
atom force field and the all atom charge set as implemented
in the program Macromodel version 5.5.42 The generalized
Born/surface area (GB/SA) method for water developed by
Still41 was used in all calculations. The number of torsion
angles allowed to vary simultaneously during each Monte
Carlo step ranged from 1 to n - 1, where n equals the total
number of rotatable bonds (n )9 in 7a and 7b, n ) 10 in 8, n
) 11 in 27, n ) 6 in 28). All torsion angles, except the N- and
C-terminal amide bond, were defined as rotatable. Conforma-
tional searches was conducted by use of the systematic
unbound multiple minimum search (SUMM) method64 in the
Batchmin program (command SPMC); 10 000 step runs were
performed, and those conformations within 12 kcal/mol of the
global minimum were kept. Two ring closure bonds, one for
each ring, were defined for the bicyclic system. Torsional
memory and geometrical preoptimization were used. Trun-
cated Newton conjugated gradient (TNCG) minimization with
a maximum of 5000 iterations was used in the conformational
search with the default derivate convergence set at a value of
0.001 (kJ /mol)/Å.
An g II An a logu e 25. The partially protected peptide resin
prepared by the procedure used for 24 was recovered in 98.4%
yield according to the weight increase. Part of the resin (126
mg, 38.8 µmol) was treated with 95% aqueous TFA and the
product was worked up as described for 24. Yield: 40.1 mg
(100%). The product was purified in two runs on a Vydac 10-
µm C18 column (1 × 25 cm) using an 80-min gradient of 10-
50% MeCN in 0.1% aqueous TFA at a flow rate of 3 mL/min
to yield 24.9 mg (33.0%) of pure 25: PDMS (MW 1034.0)
1035.1 (M + H+); amino acid analysis Asp, 0.98; Arg, 1.01;
Val, 0.98; Tyr, 1.13; His, 1.02; Phe, 0.28; Cys, nd (54% peptide).
Ack n ow led gm en t. We thank Anja J ohansson for
excellent assistance in the synthetic work, Susanna
Lindman for good collaboration concerning the in vitro
binding experiments on CHO cells, and the Swedish
Research Council for Engineering Sciences (TFR) and
the Swedish Foundation for Strategic Research (SFF)
for financial support.
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C NMR
spectra for compound 12. This material is available free of
charge via the Internet at http://pubs.acs.org.
An g II An a logu e 26. Synthesis, cleavage, and purification
were carried out as described for 24 above. The yield of peptide
resin was 242 mg (97.2%) and of crude product 59.3 mg
(75.6%). The purified material weighed 18.6 mg (15.6%):
PDMS (MW 1046.1) 1046.7 (M + H+); amino acid analysis Asp,
0.99; Arg, 1.01; Tyr, 0.91; His, 1.00; Pro, 1.00; Phe, 1.10
(overlapping with an impurity derived from Hcy); Hcy, nd (66%
peptide).
AT1 Recep tor Bin d in g Assa y on CHO Cells. CHO cells
stably expressing the rat AT1 receptor38 were grown in RPMI
1640 medium with L-glutamine, supplemented with 10% fetal
calf serum and antibiotic/antimycotic solution, in a humidified
atmosphere of 5% CO2 and 95% air. At 80% confluency, the
cells were detached from the flasks by incubation in Hanks’
balanced salt solution (HBSS) with 5 mM EDTA, collected by
centrifugation, and suspended in binding buffer consisting of
minimum essential medium with Earle’s salts, 25 mM HEPES,
GlutaMax I, 0.2% BSA, 0.02% phenanthroline, leupeptin (0.5
µg/mL), and bacitracin (0.2 mg/mL). The cells were distributed
in a 96-well plate so that each well contained about 50 000
cells. The cells were incubated with a fixed concentration of
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125I]Ang II (ca. 50 000 cpm, ca. 0.2 nM) and variable concen-
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