Tetrahydroisoquinoline-based spirocyclic lactam as a type II′ β-turn inducing peptide mimetic

Article abstract of DOI:10.1021/jo901480d

(Chemical Equation Presented) We present here spirocyclic lactam derivatives, embodying D-Phe and L-Ala amino acids as the central core and acting as tetrapeptide and hexapeptide mimetics. An efficient route for their synthesis is demonstrated by using th

Full text of DOI:10.1021/jo901480d

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Tetrahydroisoquinoline-Based Spirocyclic Lactam as a Type II0 β-Turn
Inducing Peptide Mimetic
Giordano Lesma,† Nicola Landoni,† Tullio Pilati,‡ Alessandro Sacchetti,§ and
Alessandra Silvani*,†
†
ꢀ
Dipartimento di Chimica Organica e Industriale, Universita degli Studi di Milano, via G. Venezian 21, 20133
Milano, Italy, ‡Istituto di Scienze e Tecnologie Molecolari-CNR, Via Golgi 19, 20133 Milano, Italy, and
§Politecnico di Milano, Dipartimento di Chimica, Materiali ed Ingegneria Chimica ‘Giulio Natta’,
via Mancinelli 7, 20131 Milano, Italy
alessandra.silvani@unimi.it
Received July 10, 2009
We present here spirocyclic lactam derivatives, embodying D-Phe and L-Ala amino acids as the
central core and acting as tetrapeptide and hexapeptide mimetics. An efficient route for their
synthesis is demonstrated by using the strategic combination of Seebach’s self-reproduction of
chirality chemistry and the Pictet-Spengler condensation as key steps. The conformational behavior
of peptide mimetics was investigated by molecular modeling, X-ray crystallography, NMR (solvent
and temperature dependence), IR spectroscopy, and circular dichroism. All data suggest very stable
and highly predictable type II0 β-turn conformations.
Introduction
As an example, over 100 peptide-activated G protein-
coupled receptors recognize ligands with turn structures.3
These receptors represent a large, promising, and still under-
developed source of new pharmaceutical targets for treating
human deseases or medical conditions.
Therefore, drug research is focused on scaffolds that, by
restricting the conformational freedom of the peptide chain,
provide structural stabilization if incorporated in short
linear or cyclic peptides, in order to enhance both target
binding and selectivity properties. Several reverse-turn sur-
rogates have been employed to constrain the backbone
geometry and side-chain conformations, and to so develop
potent and selective peptidomimetic therapeutic agents.4
Protein-protein and peptide-protein interactions play
key roles in most biological processes, and therefore re-
present valuable targets for drug discovery. A general
approach in the search of new chemical entities able to
interfere with these interactions is the use of small com-
pounds mimicking or inducing precise aspects of specific
peptide secondary structures.1 Among the secondary struc-
ture elements, reverse-turns are structural motifs com-
monly found in bioactive peptides that, besides being
fundamental in protein folding, play a central role as
molecular recognition elements.2
(1) For reviews, see: (a) Ohfune, Y.; Shinada, T. Eur. J. Org. Chem.
2005, 24, 5127–5143. (b) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F.
Chem. Rev. 2001, 101, 3219–3232. (c) Gibson, S. E.; Guillo, N.; Tozer,
M. J. Tetrahedron 1999, 55, 585–615. (d) Hanessian, S.; McNaughton-
Smith, G.; Lombart, H.-G.; Lubell, W. D. Tetrahedron 1997, 53, 12789–
12854.
(2) (a) Marcelino, A. M. C.; Gierasch, L. M. Biopolymers 2008, 89, 380-391
and references cited therein. (b) Ball, J. B.; Hughes, R. A.; Alewood, P. F.;
Andrews, P. R. Tetrahedron 1993, 49, 3467–3478.
(4) For selected references about reverse-turn surrogates, see: (a) Beierle,
J. M.; Horne, W. S.; van Maarseveen, J. H.; Waser, B.; Reubi, J. C.; Ghadiri,
M. R. Angew. Chem., Int. Ed. 2009, 48, 4725–4729. (b) Molteni, M.; Bellucci,
M. C.; Bigotti, S.; Mazzini, S.; Volonterio, A.; Zanda, M. Org. Biomol. Chem.
2009, 7, 2286–2296. (c) Trabocchi, A.; Sladojevich, F.; Guarna, A. Chirality
2009, 21, 584–594. (d) Arbor, S.; Kao, J.; Wu, Y.; Marshall, G. R.
Biopolymers 2008, 90, 384–393. (e) Palomo, C.; Aizpurua, J. M.; Ganboa,
I.; Benito, A.; Cuerdo, L.; Fratila, R. M.; Jimenez, A.; Loinaz, I.; Miranda,
J. I.; Pytlewska, K. R.; Micle, A.; Linden, A.; et al. Letters 2004, 6, 4443–
4446. (f ) Gutierrez-Rodriguez, M.; Garcia-Lopez, M. T.; Herranz, R.
Tetrahedron 2004, 60, 5177–5183.
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8098 J. Org. Chem. 2009, 74, 8098–8105
Published on Web 09/29/2009
DOI: 10.1021/jo901480d
r
2009 American Chemical Society

Lesma et al.
JOCArticle
FIGURE 1. Tetrahydroisoquinoline-based reverse turn mimetics.
Conformationally constrained amino acid mimics have
found widespread use in this context.5
Moreover, spectroscopic studies have shown that the high
affinity of the potent bradykinin analogue HOE 14010 for
B2 receptors is related to its high propensity to adopt a
C-terminal β-turn conformation,11 spanning the residue
Ser6-D-Tic7-Oic8-Arg9 (Oic = octahydroindole-2-carboxylic
acid). Quite recently, a modest but significant increase in
therapeutic index for a d-Tic containing analogue of anti-
microbial peptide gramicidin S (GS), has been demonstrated
generated by replacing the D-Phe residue in both the β-turn
regions of the native peptide.12
By incorporating Tic into position i þ 2 of model peptide
surrogates, we were able to generate reverse turn mimetics,
such as 1a,b and 2a,b (Figure 1), which can be considered Ac-
Ala-Phe-NHMe constrained isosteres.5 In particular, con-
formational studies performed by NMR, IR, and molecular
modeling techniques demonstrated a stable type II0 β-turn
conformation in solution for tetrapeptide analogue 2b.
Through a variation of our synthetic approach, the Tic-
derived reverse turn nucleating moiety was now moved into
position i þ 1, enabling the formation of a spirocyclic lactam
bridge to the backbone nitrogen of aminoacid at position
i þ 2.13 The obtained spiro-pyrrolo-tetrahydroisoquinoline
(SIPP) (SIPP = 2-(20-oxo-2,4-dihydro-1H-spiro[isoquino-
line-3,30-pyrrolidine]-10-yl)propanoic acid) scaffold, embo-
died in the tetrapeptide Ac-Phe-Ala-NHMe analogue 3,
adopted conformations almost ideally matching the prere-
quisites for canonical type II0 β-turn. From MM calcula-
tions, the size of the lactam ring seems to play a crucial role in
determining the favorite conformations, with the structure 3,
embodying a five-membered ring, showing the best aptitude
for β-turn.
In our ongoing program of identification of scaffolds of
low molecular weight of potential interest in drug discovery,
we recently focused on bicyclic and tricyclic frameworks
based on the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid, commonly referred to as Tic, as a common privileged
substructure.6
The Tic core, in which a carbon atom connects the
aromatic ring to the backbone amino group, is a key element
in several peptide-based drugs, since its incorporation not
only restricts the conformational freedom of the aromatic
ring but also places constraints on the peptide backbone.7
For instance, Tic is an essential feature of numerous potent,
high-affinity δ- and κ-selective opioid receptor agonists and
antagonists. They have been prepared by systematic mod-
ification of endogenous GPCRs ligands, such as enkephalin,
endomorphin, and dynorphin, thus allowing a better under-
standing of how opioid receptors function at the molecular
level.8 Also in the design and synthesis of peptidomimetics
acting as antagonists of the GPCRs ligand bradykinin, SAR
studies had revealed a pharmacophore containing a D-Tic
residue among the key features around the backbone core.9
ꢁ
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Madison, V.; Girijavallabhan, V. J. Med. Chem. 2006, 49, 567–574. (c) Wang,
W.; Cai, M.; Xiong, C.; Zhang, J.; Trivedi, D.; Hruby, V. J. Tetrahedron
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Santagada, V.; Kessler, H. J. Am. Chem. Soc. 1994, 116, 7532–7540. (b) Kyle,
D. J.; Blake, P. R.; Smithwick, D.; Green, L. M.; Martin, J. A.; Sinsko, J. A.;
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2002, 37, 379–389.
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Salvadori, S.; Trapella, C.; Balboni, G. J. Med. Chem. 2006, 49, 5640-5643 and
references cited therein. (b) Eguchi, M. Med. Res. Rev. 2004, 24, 182–212. (c)
Lazarus, L. H.; Bryant, S. D.; Cooper, P. S.; Guerrini, R.; Balboni, G.; Salvadori, S.
Drug Disc. Today 1998, 3, 284–294.
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Madami, A.; Meini, S.; Terracciano, R. J. Peptide Sci. 2001, 7, 270–283.
(13) For articles dealing with the use of spirocyclic lactams, see for
instance: (a) Bittermann, H.; Gmeiner, P. J. Org. Chem. 2006, 71, 97–102.
ꢂ
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TABLE 1. MC/EM Conformational Analysis for Tic-Based Spirocyclic
Lactamsa
n
no. of conf. <6 kcal/mol % dR < 7 A % |β| < 30° % H bond
0
1
2
3
8
3
9
19
37.5
100
89
26
37.5
100
89
21
25
100
0
0
aResults are reported as percentage of conformers which meet the
indicated requirement.
FIGURE 2. Parameters for the characterization of β-turn propen-
sity of tetrahydroisoquinoline-based spirocyclic lactams.
experimental and molecular mechanics investigations of its
conformational behavior. To demonstrate the general ap-
plicability of the strategy, we also elaborated a synthetic
pathway incorporating protected serine and arginine at the
i and i þ 3 positions, to build the hexapeptide 4, analogous of
the β-turn terminal portion of HOE 140. To simplify the
solution NMR analysis, Boc and NHMe groups were chosen
respectively as the (i) and (i þ 5) residues, so as to provide
methyl signals in the NMR high-field region. In particular,
the Boc group has been proposed as an N-terminal residue
mimic that does not affect the conformational behavior of
short peptides.14
FIGURE 3. Low-energy conformers for 3 and 4. For 4 hydrogen
atoms are omitted for clarity.
meeting the β-turn requirements decrease and intramolecu-
lar hydrogen bonds are absent. The spiro β-lactam ring (n =
0) also shows a poor aptitude to mimic a β-turn conforma-
tion. So we focused our attention on the spirocyclic γ-lactam
3. For 3, the same global minimum was found, both in vacuo
and in water, the latter being implicitly represented by a
solvation model.18 According to the definition of β-turn
types, the inspection of the dihedral angle values of the
amide backbone for the global minimum of 3 (Figure 3)
ascribes this compound to a β-turn of type II0 (see Table 2).
Subsequently, the serine and arginine fragments were
added to obtain the desired hexapeptide mimic 4. Also in
this case a conformational search was performed with the
same protocol. In vacuo, six conformations were found
within 6 kcal/mol of the global minimum, and in water 14.
The global minimum is the same both in vacuo and in water
(Figure 3) and its properties (Table 2) are still consistent with
a β-turn of type II0.
Synthesis. The target compounds were synthesized from
the key precursor aldehyde 5 (Scheme 1). Preparation of 5
was accomplished following the protocol described by
Scheidt et al., starting in our case from D-Phe.19 Using
chemistry introduced by Seebach,20 aldehyde 5 was obtained
in 41% overall yield as a single diastereoisomer, starting
from D-Phe.
Reductive amination of 5with the HCl salt of L-alanine methyl
ester, followed by heating in toluene in the presence of HOBt,
afforded pyrrolidinone 6, from which amine 7 was derived,
by removal of the Cbz protecting group. Pictet-Spengler
condensation (20% TFA, CH2Cl2) of amine 7 with formalde-
hyde afforded the tetrahydroisoquinoline 8 in moderate yield.
Results and Discussion
Modeling Studies. First of all, a computer-assisted con-
formational analysis, as a function of the dimension of the
lactam ring, was performed on Tic-based spirocyclic lactams
such as 3. The reverse turn mimicry ability was evaluated by
computing15 the main geometric features of β-turns. The
interatomic distance dR, here represented by the (CR1-CR4)
distance, should be less than 7 A, and the virtual torsion
angle β, defined by C1-CR2-CR3-N4, should be |β| < 30°
(Figure 2).
Another important feature in stabilizing the reverse turn
conformation is the presence of the characteristic hydrogen
bond C1O---HN4, that was estimated by means of the
“hydrogen bonds” function implemented in the software.16
The computational procedure consisted of an unconstrained
Monte Carlo/energy minimization conformational search
using the molecular mechanics MMFF94 force field17 in
vacuo. For each compound only conformations within 6
kcal/mol of the global minimum were kept. Results are
reported in Table 1 as percentage of conformers which meet
the requirements for a generic β-turn.
The best performance is achieved with compound 3 (n =
1), with 100% of conformers satisfying all the β-turn require-
ments. The low number of conformers within 6 kcal/mol of
the global minimum (3 conformers) is a consequence of the
high rigidity of this scaffold. The increase of the lactam ring
size produces more flexible structures with a rise of confor-
mers within 6 kcal/mol of the global minimum (9 conformers
for n = 2 and 19 for n = 3). The percentages of conformers
(18) The empirical solvation model SM.4 implemented in Spartan ’08 was
applied. This model estimates the aqueous solvation energy. See also:
Chambers, C. C.; Hawkins, G. D.; Cramer, C. J.; Truhlar, D. G. J. Phys.
Chem. A 1996, 100, 16385–16398.
(14) Shaw, R.; Perczel, A.; Fasman, G. D.; Mantsch, H. H. Int. J. Peptide
Res. 1996, 48, 71–78.
(15) Spartan’08; Wavefunction, Inc., Irvine, CA.
(16) Hydrogen bonds are defined as nonbonded contacts between a
nitrogen or oxygen and a hydrogen attached to nitrogen or oxygen, separated
by a distance ranging from 1.6 to 2.1 A and making an X-H-Y (X, Y = N, O)
angle >120°.
(19) (a) Scheidt, K. A.; Roush, W. R.; McKerrow, J. H.; Selzer, P. M.;
Hansell, E.; Rosenthal, P. J. Bioorg. Med. Chem. 1998, 6, 2477–2494. (b)
Zydowsky, T. M.; Dellaria, J. F. Jr.; Nellans, H. N. J. Org. Chem. 1988, 53,
5607–5616.
(17) Halgren, T. A. J. Comput. Chem. 1996, 17, 490–519.
(20) Seebach, D.; Fadel, A. Helv. Chim. Acta 1985, 68, 1243.
8100 J. Org. Chem. Vol. 74, No. 21, 2009

Lesma et al.
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TABLE 2. Calculated Values of Selected Geometrical Parameters for the Lowest Energy Conformers of 3 and 4 and X-ray Derived Geometrical
Parameters for 3 in the Solid State
compd
(i)C=O HN(iþ3) (A)
Φ
(iþ1); ψ(iþ1) (deg)
Φ
(iþ2); ψ(iþ2) (deg) d(CR(i) CR(iþ3)) (A) β (deg) (i)O H-N(iþ3) (deg)
3 3 3
1.957
3 3 3 3 3 3
3 (MM/MC low energy
conformera)
3 (solid state)
4 (MM/MC low energy
conformera)
II0 β-turn (ideal)
51.7; -136.4
-103.8; 41.1
5.59
-19.5
156.63
2.103
1.856
48.9; -133.7
53.3; -137.5
-89.4; 10.8
-96.6; 43.4
5.47
5.27
-11.9
-12.6
168.9
147.98
1.8-2.5
60; -120
-80; 0
<7
0
160
aThe global minimum is the same both in vacuo and in water.
SCHEME 1
SCHEME 2
In the end, compound 8 was subjected to N-acetylation to afford
acetamide 9, which was reacted with H2NMe, allowing the target
tetrapeptide analogue 3 to be obtained.
Starting from tetrahydroisoquinoline 8 (Scheme 2), BOP-
Cl mediated condensation with Boc-O-benzyl-L-serine af-
forded 10 in moderate yield. Hydrolysis of methyl ester with
LiOH to give 11, followed by BOP-Cl mediated condensa-
tion with Nω-nitro-L-arginine methyl ester hydrochloride,
gave compound 12, which was converted by treatment with
H2NMe to the desired hexapeptide 4.
2-propanol solution of the tetrapeptide mimic 3. Figure 4
shows the molecular structure of 3 in the crystal, where it
adopts a turn conformation, with an 2.103 A intramolecular
hydrogen bond between the COi oxygen atom acceptor and
the NHiþ3 amide donor (see Table 2). The hydrogen bond
directionality defined by the (i)
is almost ideal.
O
H-N(iþ3) angle of 168.9°
3 3 3
For β-turns, an additional important criterion is that the
CR(i) CR(iþ3) distance should be smaller than 7 A. The
3 3 3
X-ray crystal structure of 3 shows a CR(i) CR(iþ3) distance
3 3 3
Conformational Analysis. The results from the computa-
tional modeling studies showed that the SIPP core in both
peptide analogues 3 and 4 could serve as a turn-inducing
element and that for both model systems a β-turn (type II0) is
a viable conformation. With 3 and 4 in hand, we now
undertook a detailed conformational investigation on the
secondary structure of these model systems, by means of
X-ray crystallography and spectroscopic techniques.
X-ray Crystal Structure Determination of 3. We were able
to obtain single crystals for crystrallographic analysis from a
of 5.467 A, which nicely complies with a β-turn arrangement.
A more detailed analysis of the structural data revealed the
presence of a well-defined β-turn conformation of type II0. In
fact, in addition to the typical intramolecular hydrogen
bond, all structural criteria used to define canonical type
II0 β-turns are also satisfactory fulfilled by the peptidomi-
metic in the solid state. The X-ray outcome is remarkable,
since it highlights that an intramolecular hydrogen bond
persists also when the intermolecular packing forces have
become important, that is to say in crystal formation and,
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Lesma et al.
JOCArticle
parameter r (1 - Δδ/Δδref),24 as a further probe of β-turn
conformation. Δδ (δ(in DMSO-d6) - δ(in CDCl3)) is the
chemical shift difference for NH between solutions in two
neat solvents referred to compound 3, while Δδref is for a
reference proton with no possibility of a 10-membered
intramolecular hydrogen bond. As a reference, we have used
the NHMe amide proton in compound 13 (Scheme 3). We
observed an r value of 0.90, which is a clear evidence of
stability for the 10-membered hydrogen bond in 3.
The study of the conformational behavior of the hexapep-
tide analogue 4 was conducted in DMSO-d6, due to the
difficulty in assigning the NH signals in CDCl3. The spin
system identification and assignment of individual reso-
nances were performed by means of bidimensional NMR
techniques, and fully accomplished also in the region of NH
signals, except for NH3 (see Figure 5), whose signal overlaps
in the aromatic zone. 2D NOESY NMR data did not suggest
the close proximity of the two peptide chains, since no
interstrand correlations, but only intrastrand contacts, were
observed. As usual, the presence of intramolecular hydrogen
bonding was determined by evaluation of the chemical shift
values of the amide NH protons and their temperature
coefficients (ΔδH/ΔT). The chemical shift of 7.90 ppm and
the temperature coefficient of -2.69 ppb/deg for NH2 sup-
port a 10-membered hydrogen-bonded state also for 4, as
alreadypredictedbythecomputationalstudies(seeFigure3).
On the other hand, the chemical shift of 6.85 ppm and the
large temperature dependence (-8.03 ppb/deg) for NH1
would suggest a weak 14-membered hydrogen-bonded state
in equilibrium with a non-hydrogen-bonded conformations.
In our opinion, this behavior is due to the engagement of the
H-bond acceptor CONH3Me in an intramolecular hydrogen
bond with NH4, as strongly suggested by the chemical shift
(7.75 ppm) and by the low (-1.69 ppb/deg) temperature
coefficient of NH4. Evidently, the peculiar NH containing
side chain of arginine hampers the two peptide chains of
hexapeptide 4 to take an antiparallel alignment in a 2:2 type
β-hairpin25 arrangement, as, also in this case, neatly pre-
dicted by MM calculations, for the minimum energy con-
former (see Figure 3).
FIGURE 4. Plot of the molecular structure of compound 3, as
determined by X-ray crystallography. Atomic displacement para-
meters at the 50% probability level (hydrogen atoms omitted: see
the Experimental Section for details). β-Turned conformation and
intramolecular 10-membered H bond are shown. A coordinated
molecule of 2-propanol is also present.
even more, in the presence of a highly coordinating protic
solvent such as iPrOH.
Unfortunately, we did not succeed in obtaining crystals of
mimic 4 that were of sufficient quality for an X-ray diffrac-
tion analysis.
Since intermolecular forces in the solid phase may lead to a
change in secondary structure, solution phase studies were
also required in order to give a complete representation of
the conformation for both 3 and 4.
1H NMR Studies. Spin system identification and assign-
ment of individual resonances for peptidomimetic model 3
1
were straightforward with H-COSY. The study of confor-
mational behavior was conducted primarily in CDCl3, to
identify intramolecular hydrogen bonding.21 The involve-
ment of the NH amide proton in intramolecular hydrogen
bonding was first estimated from evaluation of its chemical
shift value and of the temperature coefficient (CDCl3), by
recording 12 spectra from 218 to 328 K with increments of
approximately 10 deg (Table 3). All data were measured at
2.0 mM concentration, that is in the absence of any notice-
able intermolecular aggregation. The downfield chemical
shift value (7.79 ppm) and the significant small temperature
coefficient (-2.75 ppb/deg) were found for the NH proton of
3, thus confirming its participation in intramolecular hydro-
gen bonding.
A supplementary indication of intramolecular hydrogen
bonding was obtained from DMSO titration studies in
CDCl3,22 indicating that the chemical shift of NH in 3 is
almost constant up to 30% of DMSO (chemical shift differ-
ence of 0.11 ppm in absolute value23). Also by switching from
the noncoordinating (CDCl3) solvent to the hydrogen bond-
acceptor solvent DMSO-d6 a negligible chemical shift dif-
ference (0.15 ppm in absolute value) was found, which
indicates a strongly hydrogen-bonded state for the amide
proton, not accessible to the solvent.
FT-IR Studies.26 The IR spectrum of a 2.0 mM solution of
3 in CHCl3 showed a unique, extensive absorption band at
3352 cm-1 for a hydrogen-bonded NH stretch, thus further
attesting the presence of a stable secondary structure for 3.
The IR spectrum of 4 exhibited an extensive absorption
band at 3322 cm-1 for the hydrogen-bonded NH stretch and
a broad band at 3438 cm-1 for the non-hydrogen-bonded
NH stretch, in accordance with the presence of both states in
this molecule.
CD Studies.27 The ability of peptidomimetic 4 to adopt a
secondary structure in solution was evaluated by CD
spectroscopy (Figure 6). The spectrum was measured in
methanol (0.2 mM) and displayed two negative minima,
one at 203 nm and a second one at 218 nm, and a negative
maximum at 209 nm. To our delight, this behavior follows
completely the same pattern with respect to well-defined
type II0 β-turn peptide backbone geometries. In fact, it has
Finally, since NH in compound 3 has two potential modes
of intramolecular hydrogen bond, that is to say 7-membered
(γ-turn) and 10-membered ring (β-turn), we evaluated the
stability of the 10-membered hydrogen bond structure by the
(21) Belvisi, L.; Gennari, C.; Mielgo, A.; Potenza, D.; Scolastico, C. Eur.
J. Org. Chem. 1999, 389–400.
(22) For all data, see the Supporting Information.
(23) For DMSO-d6/CDCl3 solutions, chemical shifts were standardized
by reference to residual proton resonance for CHD2CD3SO (2.49 ppm).
(24) Tonan, K.; Ikawa, S. Spectrochim. Acta A 2003, 59, 111–120.
ꢂ
(25) de Alba, E.; Jimenez, M. A.; Rico, M. J. Am. Chem. Soc. 1997, 119,
175–183.
(26) Vass, E.; Hollosi, M.; Besson, F.; Buchet, R. Chem. Rev. 2003, 103,
1917–1954.
(27) Cai, X.; Dass, C. Curr. Org. Chem. 2003, 7, 1841–1854.
ꢀ
8102 J. Org. Chem. Vol. 74, No. 21, 2009

Lesma et al.
JOCArticle
TABLE 3. Chemical Shifts and Temperature Coefficients of Amide Protons in 3 and 4a
ΔδNH/ΔT
(ppb/deg)
ΔδNH1/ΔT
(ppb/deg)
ΔδNH2/ΔT
(ppb/deg)
ΔδNH4/ΔT
(ppb/deg)
compd
δNH (ppm)
δNH1 (ppm)
δNH2 (ppm)
δNH4 (ppm)
3
4
7.79
-2.75
6.85
7.90
7.75
-8.03
-2.69
-1.69
aAll NMR experiments were performed with 2.0 mM solutions in CDCl3 (for 3) or DMSO-d6 (for 4). Chemical shift values (δ) reported are the values
at 298 K. Temperature coefficients were determined between 218 and 328 K for 3 and between 303 and 353 K for 4. See the SupportingInformation for all
experimental data.
central core. Both MM calculations and spectroscopic NMR,
IR, and X-ray investigations support the conclusion that the
SIPP scaffold embodied in the spirocyclic lactam 3 is a potent
and very stable type II0 β-turn inducer, with highly predictable
stereostructural properties. The turn mimic properties of 3 are
efficiently transferred to the hexapeptide mimic 4, for which a
type II0 β-turn conformation was also predicted by MM
calculations and strongly assessed with NMR, IR, and CD
FIGURE 5. Compound 4 including amide proton numbering.
experiments.
Work to illustrate the application of these novel spirocyc-
lic lactam peptidomimetics to the synthesis of β-turned
ligands for G-protein-coupled receptors is in progress in
our laboratory.
Experimental Section
General Methods. All solvents were distilled and properly
dried, when necessary, prior to use. All chemicals were pur-
chased from commercial sources and used directly, unless
indicated otherwise. All reactions were run under N2, unless
otherwise indicated. All reactions were monitored by thin layer
chromatography (TLC) on precoated silica gel 60 F254; spots
were visualized with UV light or by treatment with 1% aqueous
KMnO4 solution. Products were purified by flash chromatog-
raphy on silica gel 60 (230-400 mesh). 1H and 13C NMR spectra
were recorded with 300 and 400 MHz spectrometers. Chemical
shifts (δ) are expressed in ppm relative to TMS at δ = 0 ppm for
1H NMR and relative to CDCl3 at δ = 77.16 ppm for 13C NMR.
FIGURE 6. CD spectrum of peptidomimetic 4 (MeOH, 0.2 mM
solution).
High-resolution MS spectra were recorded with a FT-ICR
(Fourier Transform Ion Ciclotron Resonance) instrument,
equipped with an ESI source, or a standard MS instrument,
equipped with an EI source.
SCHEME 3
(2R,4S)-Benzyl 4-Benzyl-2-tert-butyl-5-oxo-4-(2-oxoethyl)-
oxazolidine-3-carboxylate, 6. To a suspension of 529 (954 mg,
2.33 mmol), NaOAc (400 mg, 4.88 mmol), and 4 A molecular
sieves (2.60 g) in MeOH (50 mL) was added the hydrochloride
salt of alanine methyl ester (374 mg, 2.69 mmol). After 45 min,
NaCNBH3 (305 mg, 4.85 mmol) was added and the reaction
mixture was stirred at 25 °C overnight. Then, the suspension was
filtered and the filtrate was acidified to pH 1 with 1 N aq HCl
and stirred for 20 min. The solution was basified to pH 9 with
saturated aq NaHCO3 and then extracted with EtOAc (4 ꢀ
50 mL). The combined organic extracts were washed with
brine, dried over Na2SO4, filtered, and concentrated to yield a
yellow oil. This oil was dissolved in toluene (30 mL), and HOBt
(310 mg, 2.30 mmol) was added. The mixture was then heated
at reflux for 16 h then cooled, and the toluene was removed
in vacuo. The residue was taken up in EtOAc (50 mL) and
washed with saturated aq NaHCO3 (3 ꢀ 50 mL) and brine, then
dried over Na2SO4, filtered, and concentrated to dryness to
yield a crude yellow oil. Purification of the crude product by
flash chromatography (hexane/EtOAc 1:3) afforded lactam 6
been demonstrated that two distinct types of CD spectra are
associated with two of the many possible turn geometries,
that is to say the type II and II0 β-turn.28 Since the
phenylalanyl side-chain chromophore makes a negligible
contribution to the CD spectra of peptidomimetics in the
195-240-nm range, we can conclude that also hexapeptide
analogue 4 adopts a type II0 β-turn conformation.
Conclusions
Inthispaper, wewereabletoestablish anefficient approach
to a novel type of tetrapeptide and hexapeptide mimics,
embodying a constrained Phe-Ala dipeptide analogue as the
(646 mg, 67%) as a clear oil. Rf 0.39 (hexane/EtOAc 1:1). [R]25
D
þ13.3 (c 1.0, CHCl3). 1H NMR (400 MHz, CDC13, δ)
(28) Bush, C. A.; Sarkar, S. K.; Kopple, K. D. Biochemistry 1978, 17,
4951–4954.
(29) Compound 5 was prepared according to ref 17. 1H and 13C NMR
spectra are in accordance with literature. [R]25D þ19.6 (c 1.0, CHCl3).
J. Org. Chem. Vol. 74, No. 21, 2009 8103

Lesma et al.
JOCArticle
7.40-7.19 (m, 10H), 5.65 (br, s, 1H), 5.17 (d, AB system, J =
12.2 Hz, 1H), 5.12 (d, AB system, J = 12.2 Hz, 1H), 4.78 (q, J =
7.4 Hz, 1H), 3.71 (s, 3H), 3.28 (d, AB system, J = 12.6 Hz, 1H),
3.15 (t, J = 9.4 Hz, 1H), 3.07 (d, AB system, J = 12.6 Hz, 1H),
2.75-2.68 (m, 1H), 2.53-2.46 (m, 1H), 2.28 (dd, J = 16.6, 8.6
Hz, 1H), 1.07 (d, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDC13,
δ) 173.3, 171.4, 155.0, 136.4, 135.2, 130.1-127.2 (10C), 66.6,
61.5, 52.4, 49.4, 42.5, 41.0, 31.2, 14.1. HRMS (EI) calcd for
C23H26N2O5 410.1842, found 410.1837.
(S )-Methyl 2-((S)-3-Amino-3-benzyl-2-oxopyrrolidin-1-yl)-
propanoate, 7. To a solution of 6 (300 mg, 0.73 mmol) in 1,4-
dioxane (15 mL) was added 10% Pd/C (30 mg). After thor-
oughly flushing the flask with N2, a hydrogen atmosphere
was introduced. After 17 h, the reaction was filtered through
Celite, then concentrated in vacuo to yield a light-yellow oil.
0.26 mmol) was dissolved in anhydrous CH2Cl2 (2 mL) at 0 °C.
Et3N (40 μL, 0.29 mmol) and BOPCl (1.5 equiv, 90 mg, 0.35
mmol) were added and the reaction mixture was stirred for
40 min. Keeping the mixture at 0 °C, a solution of compound 8
(68 mg, 1.26 mmol) in anhydrous CH2Cl2 (2 mL) and Et3N
(40 μL, 0.29 mmol) was slowly added. The reaction mixture was
allowed to go up to room temperature and stirred for 24 h. The
reaction was quenched with H2O (4 mL) and the organic layer
was washed with 5% aq H3PO4 (2 ꢀ 4 mL), 5% aq NaHCO3 (2 ꢀ
4 mL), and water (4 mL), dried over Na2SO4, and filtered, then
the solvent was removed in vacuo. Purification by flash chro-
matography (hexane/EtOAc 1:1) gave compound 10 (57 mg,
43%) as a colorless oil. Rf 0.70 (EtOAc/MeOH 9:1). [R]25D -8.5
(c 1.0, CHCl3). 1H NMR (400 MHz, DMSO, 100 °C, 1:1 mixture
of two conformers, δ) 7.40-7.18 (m, 9H), 6.49 (br, d, J = 7.1 Hz,
0.5H), 6.16 (br, d, J = 7.1 Hz, 0.5H), 4.93-4.77 (m, 2H),
4.61-4.44 (m, 4H), 3.75-3.58 (m, 2H), 3.69 (s, 3H), 3.51-3.34
(m, 2H), 3.03-2.98 (m, 1H), 2.83 (dd, J = 14.6, 1.6 Hz, 1H),
2.23-2.06 (m, 1H), 1.61-1.54 (m, 1H), 1.44 (s, 4.5H), 1.42-1.39
(m, 7.5H). 13C NMR (100 MHz, CDC13, 1:1 mixture of two
conformers, δ) 174.1 and 173.8, 172.1, 170.3 and 170.1, 155.7,
138.8 and 138.3, 135.2 and 135.1, 134.8, 129.2-126.9 (9C), 80.4,
74.0, 72.4, 71.7, 70.5 and 70.4, 64.9 and 64.8, 52.9, 51.4 and 51.2,
48.1 and 47.7, 41.4 and 41.1, 37.7 and 37.5, 48.9, 29.7 and 29.5,
29.0, 15.3. HRMS (EI) calcd for C31H39N3O7 565.2788, found
565.2779.
Amine 7 (191 mg, 95%) was used without further purification.
1
Rf 0.35 (hexane/EtOAc 1:2). [R]25 -11.1 (c 1.0, CHCl3). H
D
NMR (400 MHz, CDC13, δ) 7.32-7.20 (m, 5H), 4.80 (q, J =
7.5 Hz, 1H), 3.70 (s, 3H), 3.16 (td, J = 9.2, 2.8 Hz, 1H), 2.99 (d,
AB system, J = 13.0 Hz, 1H), 2.81 (d, AB system, J = 13.0 Hz,
1H), 2.48 (dd, J = 16.9, 7.8 Hz, 1H), 2.32-2.24 (m, 1H), 1.90 (m,
3H), 1.13 (d, 7.5 Hz, 3H). 13C NMR (100 MHz, CDC13, δ)
177.4, 171.8, 136.2, 130.1 (2C), 128.3 (2C), 127.0, 60.5, 52.3,
49.4, 44.7, 39.7, 31.5, 14.3. HRMS (EI) calcd for C15H20N2O3
276.1474, found 276.1481.
(S )-Methyl 2-((S)-20-Oxo-2,4-dihydro-1H-spiro[isoquinoline-
3,30-pyrrolidine]-10-yl)propanoate, 8. To a solution of 7 (700 mg,
2.53 mmol) in CH2Cl2 (14 mL) were added (CH2O)n (137 mg,
4.56 mmol) and TFA (6 mL). After 20 h at room temperature,
saturated aq NaHCO3 was slowly added until pH 8. The layers
were separated and the aqueous layer was extracted with CH2Cl2
(2 ꢀ 15 mL). The combined organic extracts were dried over
Na2SO4, filtered, and concentrated in vacuo. The residue was
purified by flash chromatography (EtOAc/MeOH 19:1) to give
(S)-2-((S)-2-((S)-3-(Benzyloxy)-2-(tert-butoxycarbonylamino)-
propanoyl)-20-oxo-2,4-dihydro-1H-spiro[isoquinoline-3,30-pyrro-
lidine]-10-yl)propanoic Acid, 11. To a solution of 10 (50 mg, 0.09
mmol) in THF (1 mL) at 0 °C was added 1 M LiOH (100 μL, 0.10
mmol) slowly. After 3 h, the solvent was concentrated in vacuo
and 5% H3PO4 aq solution was added until the solution was at
pH 3. The aqueous phase was extracted with ethyl acetate (2 ꢀ
30 mL). The organic layer was dried over Na2SO4 and the
solvent was removed under reduced pressure affording pure
acid 11 (48 mg, 99%) as a foam. The compound was used
without purification. Rf 0.09 (hexane/EtOAc 1:2). [R]25D -40.9
(c 1.6, CHCl3). 1H NMR (400 MHz, CDC13, 2:1 mixture of two
conformers, δ) 7.42-6.91 (m, 9H), 5.62 (d, J = 8.6 Hz, 0.33H),
5.51 (d, J = 8.6 Hz, 0.67H), 5.18-5.05 (m, 1.33H), 5.02-4.92
(m, 1.33H), 4.65-4.53 (m, 2H), 4.48 (d, AB system, J = 11.7 Hz,
0.67H), 4.39 (d, AB system, J = 11.7 Hz, 0.67H), 3.82-3.71 (m,
1.67H), 3.55-3.31 (m, 3.33H), 3.28 (d, AB system, J = 14.6 Hz,
0.67H), 3.26 (d, AB system, J = 14.6 Hz, 0.33H), 2.74 (d, AB
system, J = 14.6 Hz, 0.33H), 2.72 (d, AB system, J = 14.6 Hz,
0.67H), 2.21-1.99 (m, 1H), 1.90-1.72 (m, 1H), 1.52 (d, J = 7.3
399 mg (48%) of 8, as an oil. Rf 0.58 (EtOAc/MeOH 4:1). [R]25
D
-13.8 (c 1.0, CHCl3). 1H NMR (300 MHz, CDC13, δ) 7.15-7.02
(m, 4H), 4.85 (q, J = 7.4 Hz, 1H), 4.13 (d, AB system, J = 17.3
Hz, 1H), 4.04 (d, AB system, J = 17.3 Hz, 1H), 3.72 (s, 3H), 3.56
(td, J = 14.0, 3.4 Hz, 1H), 3.48 (dt, J = 14.0, 7.5 Hz, 1H), 2.99 (d,
AB system, J = 16.2 Hz, 1H), 2.61 (d, AB system, J = 16.2 Hz,
1H), 2.23-2.10 (m, 1H), 2.03-1.93 (m, 2H), 1.46 (d, 7.4 Hz, 3H).
13C NMR (100 MHz, CDC13, δ) 175.9, 171.7, 134.6, 132.6, 129.7,
128.3, 126.3, 125.8, 59.2, 52.4, 49.8, 44.2, 40.8, 34.1, 30.4, 14.9.
HRMS (EI) calcd for C16H20N2O3 288.1474, found 288.1466.
(S)-Methyl 2-((S)-2-Acetyl-20-oxo-2,4-dihydro-1H-spiro[isoquino-
line-3,30-pyrrolidine]-10-yl)propanoate, 9. Compound 8 (50 mg,
0.17 mmol) was dissolved in CH2Cl2 (2 mL) and DIPEA (45 μL,
0.26 mmol) was added. The solution was then cooled to 0 °C and
acetic anhydride (24 μL, 0.26 mmol) was slowly added. After the
addition, the mixture was stirred at room temperature for 12 h. Then,
the reaction mixture was poured into ice water and extracted with
CH2Cl2 and the organic solution was washed with 5% aq NaHCO3.
The organic phase was then dried over Na2SO4, and the solvent was
removed under reduced pressure to afford 9(56mg, 98%yield) asan
oil. The compound was used without further purification. Rf 0.56
Hz, 2H), 1.51 (d, J = 7.3 Hz, 1H), 1.47 (s, 6H), 1.44 (s, 3H). 13
C
NMR (100 MHz, CDC13, 2:1 mixture of two conformers, δ)
173.3 (major) and 173.1 (minor), 171.9 (minor) and 171.8
(major), 170.7, 155.0, 137.8 (minor) and 137.3 (major), 133.9
(minor) and 133.7 (major), 133.5, 128.3-126.4 (9C), 80.2
(major) and 80.1 (minor), 73.5, 71.5, 70.6, 64.4 (minor) and
64.3 (major), 51.6, 50.6, 47.7, 47.3, 40.2, 37.4 (minor) and 37.2
(major), 30.0 (minor) and 29.8 (major), 28.4, 14.2. HRMS (EI)
calcd for C30H37N3O7 551.2632, found 551.2647.
(S)-Methyl 2-((S)-2-((S)-2-((S)-3-(Benzyloxy)-2-(tert-butoxy-
carbonylamino)propanoyl)-20-oxo-2,4-dihydro-1H-spiro[isoquino-
line-3,30-pyrrolidine]-10-yl)propanamido)-5-((E)-2-nitroguanidino)-
pentanoate, 12. The acid 11 (28 mg, 0.05 mmol) was dissolved in
anhydrous CH2Cl2 (1 mL) at 0 °C. Et3N (10 μL, 0.07 mmol) and
BOPCl (1.5 equiv, 20 mg, 0.08 mmol) were added and the reaction
mixture was stirred for 40 min. Keeping the mixture at 0 °C, Nω-
nitro-L-arginine methyl ester HCl (15 mg, 0.06 mmol) previously
dissolved in anhydrous CH2Cl2 (1 mL) and Et3N (10 μL, 0.07
mmol) was slowly added. The reaction mixture was allowed to go
up to room temperature and stirred for 24 h. The reaction was
quenched with H2O (4 mL) and the organic layer was washed with
5% aq H3PO4 (2 ꢀ 4 mL), saturated aqueous solution of NaHCO3
(EtOAc/MeOH 8:1). [R]25 -20.5 (c 0.9, CHCl3). 1H NMR (400
D
MHz, CDCl3, δ) 7.34-7.22 (m, 4H), 4.76 (q, J = 7.4 Hz, 1H), 4.65
(d, AB system, J = 14.2 Hz, 1H), 4.57 (d, AB system, J = 14.2 Hz,
1H), 3.79 (s, 3H), 3.58 (td, J = 9.9, 2.2 Hz, 1H), 3.35 (q, J = 8.5 Hz,
1H), 3.19 (d, AB system, J = 14.6 Hz, 1H), 2.69 (d, AB system, J =
14.6 Hz, 1H), 2.31-2.23 (m, 1H), 2.25 (s, 3H), 1.68-1.62 (m, 1H),
1.50 (d, 7.4 Hz, 3H). 13C NMR (100 MHz, CDC13, δ) 174.3, 172.1,
170.0, 135.4, 135.0, 128.7, 128.6, 127.8, 126.6, 64.2, 52.9, 51.3, 48.9,
41.4, 37.9, 30.0, 23.7, 15.3. HRMS (EI) calcd for C18H22N2O4
330.1580, found 330.1586.
(S)-Methyl 2-((S)-2-((S)-3-(Benzyloxy)-2-(tert-butoxycarbonyl-
amino)propanoyl)-20-oxo-2,4-dihydro-1H-spiro[isoquinoline-3,30-
pyrrolidine]-10-yl)propanoate, 10. Boc-O-benzyl serine (77 mg,
8104 J. Org. Chem. Vol. 74, No. 21, 2009

Lesma et al.
JOCArticle
(2 ꢀ 4 mL), and water (4 mL), dried over Na2SO4, and filtered,
then the solvent was removed in vacuo. Purification by column
chromatography (EtOAc) gave compound 12 (17 mg, 45%) as a
mmol) was dissolved at room temperature in a solution of methyl
amine in EtOH (5 mL, 8.03 M). The reaction mixture was stirred
for 12h andevaporated to give 13 (21 mg, 95%) asa foam. Rf 0.63
(EtOAc/MeOH 4:1). [R]25D -26.9 (c 0.4, CHCl3). 1H NMR (400
MHz, CDCl3, δ) 7.28-7.06 (m, 4H), 6.45 (br, s, 1H), 4.73 (q, J =
7.2 Hz, 1H), 4.14 (d, AB system, J = 17.5 Hz, 1H), 4.06 (d, AB
system, J = 17.5 Hz, 1H), 3.47-3.37 (m, 2H), 3.06 (d, AB system,
J = 16.3 Hz, 1H), 2.80 (d, J = 4.7 Hz, 3H), 2.64 (d, AB system,
J = 16.3 Hz, 1H), 2.19-2.10 (m, 1H), 2.10-1.96 (m, 2H), 1.45 (d,
7.2 Hz, 3H). 13C NMR (100 MHz, CDC13, δ) 176.9, 171.3, 135.1,
133.1, 130.4, 127.0 (2C), 126.4, 60.0, 51.3, 44.8, 41.1, 34.6, 30.6,
26.9, 14.3. HRMS (EI) calcd for C16H21N3O2 287.1634, found
287.1628.
foamy oil. Rf 0.64 (EtOAc/MeOH 9:1). [R]25
-42.8
D
(c 0.7, CHCl3). 1H NMR (400 MHz, CDC13, 3:2 mixture of two
conformers, δ) 8.30 (br, s, 1H), 8.13 (br, s, 0.6H), 8.04 (br, s, 0.4H),
7.45-6.91 (m, 9H), 5.93 (br, d, J = 7.2 Hz, 0.6H), 5.64 (br, d, J =
7.2 Hz, 0.4H), 5.18-4.95 (m, 2H), 4.94-4.79 (m, 2H), 4.74-4.53
(m, 3H), 4.50 (d, AB system, J = 11.8 Hz, 1H), 4.41 (d, AB system,
J =11.8Hz, 1H), 3.76(s, 1.8H), 3.80-3.71 (m, 1H), 3.69 (s, 1.2H),
3.53 (t, J = 8.8 Hz, 1H), 3.48-3.37 (m, 1H), 3.36-3.18 (m, 2H),
3.28 (d, AB system, J = 14.6 Hz, 1H), 2.77-2.71 (m, 1H), 2.72 (d,
AB system, J = 14.6 Hz, 1H), 2.21-1.99 (m, 1H), 1.90-1.72 (m,
1H), 1.52 (d, J = 7.3 Hz, 3H), 1.47 (s, 9H). 13C NMR (100 MHz,
CDC13, 3:2 mixture of two conformers, δ) 173.5, 171.8, 171.5,
170.4, 159.4, 155.6 (minor) and 155.3 (major), 137.4, 134.0, 133.5,
128.3-127.2 (8C), 126.3, 79.9, 73.4 (2C), 71.8, 70.2, 64.2, 52.6
(major) and 52.4 (minor), 52.1, 50.7, 47.4 (minor) and 47.2
(major), 40.3 (minor) and 39.9 (major), 37.8, 29.6, 29.2, 28.4
(3C), 24.8, 13.8. HRMS (EI) calcd for C37H50N8O10 766.3650,
found 766.3671.
Supporting Information Available: Detailed experimental
methods for compound 3 (from 9) and for compound 4 (from
12), crystal data for compound 3 (also in CIF format), NMR
spectra of compounds 3-4 and 6-13, VT 1H NMR data for 3
and 4, DMSO-d6 titration 1H NMR data for 3, IR spectra for 3
and 4, and computational data from conformational analysis
for 3 and 4. This material is available free of charge via the
Internet at http://pubs.acs.org.
(S)-N-Methyl-2-((S)-20-oxo-2,4-dihydro-1H-spiro[isoquinoline-
3,30-pyrrolidine]-10-yl)propanamide, 13. Compound 8 (22 mg, 0.08
J. Org. Chem. Vol. 74, No. 21, 2009 8105