Tetrahydro-β-carboline-based spirocyclic lactam as type II′ β-turn: Application to the synthesis and biological evaluation of somatostatine mimetics

Article abstract of DOI:10.1021/jo302737j

The synthesis of novel spirocyclic lactams, embodying d-tryptophan (Trp) amino acid as the central core and acting as peptidomimetics, is presented. It relies on the strategic combination of Seebach's self-reproduction of chirality chemistry and Pictet-Spengler condensation as key steps. Investigation of the conformational behavior by molecular modeling, X-ray crystallography, and NMR and IR spectroscopies suggests very stable and highly predictable type II′ β-turn conformations for all compounds. Relying on this feature, we also pursued their application to two potential mimetics of the hormone somatostatin, a pharmaceutically relevant natural peptide, which contains a Trp-based type II′ β-turn pharmacophore.

Full text of DOI:10.1021/jo302737j

Article
pubs.acs.org/joc
Tetrahydro-β-carboline-Based Spirocyclic Lactam as Type II′ β‑Turn:
Application to the Synthesis and Biological Evaluation of
Somatostatine Mimetics
Giordano Lesma,† Roberto Cecchi,‡ Alfredo Cagnotto,§ Marco Gobbi,§ Fiorella Meneghetti,∥
Manuele Musolino,*,† Alessandro Sacchetti,⊥ and Alessandra Silvani*,†
†Dipartimento di Chimica, Universita
̀
degli Studi di Milano, via C. Golgi 19, 20133 Milano, Italy
‡Sanofi-aventis, Centro Ricerche Sanofi-Midy, Via G. Sbodio 2, 20134 Milano, Italy
§Istituto di Ricerche Farmacologiche “Mario Negri”, Via La Masa 19, 20156 Milano, Italy
∥Dipartimento di Scienze Farmaceutiche, Universita
̀
degli Studi di Milano, via L. Mangiagalli 25, 20133 Milano, Italy
⊥Dipartimento di Chimica, Materiali ed Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, via Mancinelli 7, 20131 Milano,
Italy
S
* Supporting Information
ABSTRACT: The synthesis of novel spirocyclic lactams, embodying D-tryptophan (Trp) amino acid as the central core and
acting as peptidomimetics, is presented. It relies on the strategic combination of Seebach’s self-reproduction of chirality chemistry
and Pictet−Spengler condensation as key steps. Investigation of the conformational behavior by molecular modeling, X-ray
crystallography, and NMR and IR spectroscopies suggests very stable and highly predictable type II′ β-turn conformations for all
compounds. Relying on this feature, we also pursued their application to two potential mimetics of the hormone somatostatin, a
pharmaceutically relevant natural peptide, which contains a Trp-based type II′ β-turn pharmacophore.
molecular framework unique 3D properties that are relevant to
the field of drug discovery.3
INTRODUCTION
■
The conformation of a peptide is crucial for its biological
activity. Turn structures in general have received particular
attention because they play an important role in biological
recognition and signal transduction processes. Many naturally
occurring oligopeptides have been proposed to adopt turns in
their bioactive conformation. As an example, over 100 peptide-
activated G protein-coupled receptors (GPCRs) bind ligands
with a turn structure.1
The development of privileged molecular scaffolds efficiently
mimicking reverse turn motifs has attracted remarkable interest
when structural constraints are exploited to increase both
binding and selectivity. One of the successful approaches to
restrict peptide conformation is the introduction of side-chain-
restrained amino acids.2 For instance, disubstitution in the α
position of an α-amino acid leads to a conformational
constraint and a stereochemically stable quaternary carbon
center. In this context, spirocyclic scaffolds are able to provide,
upon the attachment of appropriate functional groups, useful
high-affinity ligands. Indeed, the polysubstituted central atom
common to the rings of spiro compounds confers on the overall
In our ongoing program aimed at identifying peptidomimetic
scaffolds of low molecular weight, we recently focused on
tetrahydroisoquinoline-based spirocyclic lactams4 and spiropi-
peridine-3,3′-oxindole scaffolds5 as, respectively, type II′ and II
β-turn inducing moieties, for which highly predictable stereo-
structural properties could be demonstrated by means of
molecular modeling calculations and spectroscopic studies.
Since many natural peptides containing tryptophan (Trp)-
based pharmacophores exhibit a wide range of important
bioactivities, and so also structurally related 1,2,3,4-tetrahydro-
β-carboline (THBC)-based compounds, we have recently
moved our attention to conformationally constrained spirocy-
clic Trp analogues6 in order to develop new reverse-turn
nucleating moieties able to be inserted into pharmacologically
relevant peptidomimetic compounds.
Among peptides sharing a Trp-containing β-turn motif in
which the Trp residue is critical for binding, we looked at the
Received: December 27, 2012
Published: February 14, 2013
© 2013 American Chemical Society
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Figure 1. Molecular formulas of somatostatin (SRIF-14, 1) and Sandostatin (octreotide, 2).
hormone peptide somatostatin (somatotropin release-inhibiting
factor, SRIF).7 Somatostatin is normally expressed as a
tetradecapeptide (SRIF-14, 1, Figure 1) or an N-terminally
extended form (SRIF-28). It appears in several organ systems,
such as the central nervous system, the hypothalamo-pituitary
system, the gastrointestinal tract, the exocrine and endocrine
pancreas, and the immune system. In these different systems,
somatostatin acts as a neuromodulator and a neurotransmitter
as well as a potent inhibitor of various secretory processes and
cell proliferation.8
spirocyclic lactam 3 (Figure 2) as a type-II′ β-turn model
compound, the application of its core structure to the synthesis
Somatostatin action is mediated by specific, high-affinity
somatostatin receptors located on the plasma membrane of the
target cells. To date, five human somatostatin receptor subtypes
(sst1, sst2, sst3, sst4, and sst5) have been cloned and
characterized. These subtypes belong to a superfamily of
GPCRs that can functionally couple to various intracellular
effector systems.9 The somatostatin receptors, which are
overexpressed in a majority of neuroendocrine tumors, also
represent the first and best example of targets for radiopeptide-
based imaging and radionuclide therapy. Radiolabeled
somatostatin analogues permit the localization and staging of
neuroendocrine tumors that express the appropriate somatos-
tatin receptors.10
The low bioavailability and poor pharmacokinetics of
somatostatin have led to interest in peptidomimetic analogues
which may be better drug candidates.11 Several hexa- and
octapeptide analogues have been developed, including the drug
Sandostatin (octreotide 2,12 a cyclic octapeptide analogue)
which is clinically used for the treatment of endocrine tumors
and acromegaly.13
Somatostatin and octreotide are thought to interact with the
sst1−5 receptors mainly by inserting a β-turn substructure,
carrying a lysine (Lys) and a Trp side chain, into a pocket of
the G protein-coupled somatostatin receptor.14 SRIF peptidic
structure−activity relationship (SAR) studies clearly indicated
that the core residues Trp8 and Lys9 (numbering of the
residues follows that of native SRIF) are the essential binding
sites for all somatostatin receptors,15 whereas Phe6 is
specifically important for activation of subtype sst4, which has
been recently recognized as an ideal therapeutic target for
Alzheimer’s disease.16 With regard to the bioactive conforma-
tion of SRIF and analogues, an important step forward was
taken from the finding that a type-II′ β-turn is present in the D-
Trp8-SRIF series, in which D-Trp8 and Lys9 are located at the i
+1 and i+2 position, respectively.17
Figure 2. 1,2,3,4-Tetrahydro-β-carboline (THBC)-based spirocyclic
lactam 3 and somatostatin mimetic 4.
of the somatostatin mimetic 4 and of the closely related 21 (see
later), and the result of biological evaluation of the latter
compounds.
RESULTS AND DISCUSSION
■
In target 3, the THBC-derived reverse turn nucleating moiety is
placed at position i+1 of a model β-turn, and it enables the
formation of a spirocyclic lactam bridge to the backbone
nitrogen of L-Ala amino acid at the i+2 position. The resulting
scaffold can so be considered a tetrapeptide Ac-Trp-Ala-NHMe
analogue, and according to a preliminary computational
evaluation, it promises to adopt conformations almost ideally
matching the prerequisites for canonical type II′ β-turn. From
MM calculations, 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. The computed main geometric features of
β-turns (Figure 3), such as the interatomic distance dα (Cα1−
We report here the preparation and structural character-
ization of the 1,2,3,4-tetrahydro-β-carboline (THBC)-based
Figure 3. Parameters for the characterization of β-turn propensity of
1,2,3,4-tetrahydro-β-carboline (THBC)-based spirocyclic lactams.
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a
Table 1. Results from Conformational Analysis
no. of conf < 6 (kcal/mol)
% dα < 7 Å
% |β| < 30
% H bond β-turn (type B)
% H bond γ-turn (type A)
n = 0
n = 1
n = 2
15
13
9
13 (6.20 Å)
38 (5.62 Å)
77 (6.27 Å)
7 (51.7°)
38 (−16.6°)
66 (−9.9°)
0 (absent)
15 (present)
0 (absent)
40 (present)
46 (absent)
77 (present)
a
Results are reported as the percentage of conformers that meet the indicated requirement. Values for the global minimum are reported in
parentheses.
Table 2. Alignment Scores Values for the Superimposition of the Spirocyclic Lactams Amide Backbones (Global Minimum for n
= 0, 1, and 2) with Standard Classical β-Turn Types
n =
I
I′
II
II′
III
III′
V
V′
0
1
2
0.88
0.87
0.84
<0.7
0.81
0.92
0.88
0.86
0.97
0.94
0.88
0.83
0.82
<0.7
<0.7
0.93
0.90
0.86
0.88
0.84
0.80
0.75
<0.7
0.78
Cα4), the virtual torsion angle β (C1−Cα2−Cα3−N4), and the
possible presence of the hydrogen bond C1O---HN4, are
reported in Table 1 for different sizes of the lactam ring.
The highest percentages of conformers satisfying the β-turn
dα and β requirements are found for the model compound with
n = 2, followed by the one with n = 1 (compound 3). The β-
lactam ring (n = 0) seems to be unsuited to induce a β-turn
conformation. The analysis further revealed the presence of two
different H bonds, a seven-membered ring H bond (type A in
Figure 3) resembling the γ-turn, and the typical 10-membered
ring H bond (type B) of the β-turn. Only compound 3 is able
to arrange an H bond of type B, whereas for the two other
model compounds, a γ-turn around the i+1−i+3 residues is
stabilized through the formation of H bond of type A.
The ability to mimic specific β-turns was also evaluated by
superimposing the amide backbone of the global minima for n
= 0, 1, and 2 (further optimized by DFT calculations at the
B3LYP-6-31G* level) with standard classical I−V′ β-turn types.
The results are reported in Table 2 as alignment scores values
ranging from 0 to 1, with 1 being the perfect alignment.
The γ-lactam ring (n = 1, compound 3) produces a very
suitable mimic for a type II′ β-turn (0.97 score, see Figure 4,
left), followed by the δ-lactam ring (n = 2, 0.94 score), the
difference being due to the lack of the typical ten-membered
ring H bond in the latter model compound.
On the basis of predictions from computational studies, we
then focused our attention to the synthesis of compounds 3
and 4, for which the common intermediate 10 was envisioned
(Scheme 1). The synthesis of aldehyde 10 was carried out from
N-Boc-D-tryptophan methyl ester adapting, as guideline, the
protocol described from Scheidt and co-workers,18 according to
the concept of self-reproduction of chirality of Seebach.19
Since, in our hands, application of Seebach’s chemistry
proved to be rather sluggish on NH-indole-containing
tryptophan, we pursued the synthesis of the suited N-TIPS-
protected derivative 7 as a valuable substrate for the α-
alkylation step. Starting from N-Boc-D-tryptophan methyl ester,
introduction of the TIPS protecting group20 afforded 5, on
which selective removal of the Boc to give primary amine 6 was
cleanly and mildly effected by means of SnCl4 Lewis acid.21 The
hydrolysis of methyl ester (1 M solution of LiOH) afforded N-
TIPS-D-trp 7, on which condensation with pivalaldehyde,
followed by treatment with benzyl chloroformate, allowed us to
obtain the desired oxazolidinone derivative in 76% overall yield,
as the essentially unique diastereoisomer 8 (dr >99%, as from
1H NMR spectroscopy). Alkylation of 8, by reaction with
KHMDS at −78 °C and allyl bromide, afforded the α-
quaternary Trp-derivative as the single expected diaster-
eoisomer 9, whose stereochemistry was also verified by the
NOESY NMR experiment (see the Supporting Information).
Because of the proven facility in the oxidation of the indole
ring under ozone also at low temperature, we chose to achieve
the aldehyde 10 by a two-step protocol. Treatment of 9 with
OsO4 and NMO afforded a diasteroisomeric mixture of diols
which was reacted, without purification, with an aqueous
solution of NaIO4 in MeOH to give 10 in 90% overall yield.22
With the key aldehyde 10 in hands, we pursued the synthesis
of the spirocyclic lactam 3 (Scheme 2).
The reductive amination of 10 with the HCl salt of L-alanine
methyl ester and sodium cyanoborohydride in MeOH, followed
by heating in toluene in presence of HOBT, generated the
unique pyrrolidinone 11 in 70% yield. The cleavage of the Cbz
protecting group by catalytic hydrogenation and of TIPS by
means of a 1 M solution of TBAF in THF afforded 12 in 82%
overall yield. Amine 12 went through a mild acidic (TFA 1eq)
Pictet−Spengler reaction with an aqueous solution of form-
aldehyde in MeOH, affording carboline 13 in 66% yield. The
target compound 3 was finally achieved in 71% overall yield by
conversion of methyl ester 13 into methyl amide 14, followed
by acylation with acetic anhydride and crystallization from
EtOAc/MeOH.
Figure 4. Superimposition of the amide backbone of the global
minimum of compounds 3 (a, RMSD = 0.325 Å) and 4 (b, RMSD =
0.378 Å) with a type II′ β-turn.
Having selected structure 3, embodying a five-membered
ring, as the more promising type-II′ β-turn model compound,
we also provided a computational evaluation of somatostatin
mimetic 4, which contains pharmacophore L-Lys and L-Phe
amino acids. Also in this case a conformational search was
performed with the same protocol. The global minimum
(Figure 4, right) and its properties (see the Supporting
Information) are still consistent with a β-turn of type II′.
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Scheme 1. Synthesis of the Aldehyde Intermediate 10
Scheme 2. Synthesis of the 1,2,3,4-Tetrahydro-β-carboline (THBC)-Based Spirocyclic Lactam 3
Scheme 3. Synthesis of the Intermediate Methyl Amide 18
Starting from the same aldehyde 10, reductive amination
with the HCl salt of Nε-Boc-L-lysine methyl ester gave as well
the unique pyrrolidinone 15 in 71% yield (Scheme 3). The
cleavage of both the Cbz and TIPS protecting groups to give
amine 16, followed by Pictet−Spengler reaction, afforded
smoothly carboline 17. Probably for a major steric congestion
with respect to compound 13, the conversion of methyl ester
17 into methyl amide 18 required a 8 M solution of NH2Me in
EtOH to afford 18 in similar yield. The compound 4 was
achieved in 43% overall yield as trifluoroacetate salt by means
of HATU-mediated condensation with N-Ac-L-phe-OH to give
19, followed by treatment with TFA in CH2Cl2 (Scheme 4).
Additionally, starting from 18, HOAt/HATU-mediated con-
densation with N-Ac-L-phe-L-phe-OH, followed by treatment
with TFA, allowed us to achieve (20% overall yield) the
pentapeptide mimetic 21 for biological evaluation.
With model compound 3 and Nε-protected mimic 19 in
hand, we undertook a detailed conformational investigation on
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Scheme 4. Synthesis of the Somatostatin Mimetics 4 and 21
their secondary structure in the solid state by means of X-ray
crystallography for 3 and in solution by means of spectroscopic
techniques for both 3 and 19.
Crystals of 3 were obtained from a methanol/acetone
solution 1:1 at room temperature as white prisms. The
crystallographic structure of lactam 3 is represented in Figure
5. In order to confirm the complete stereocontrol of our
angle. The values of the distance C16−C20 of 5.69(1)Å and of
the virtual torsion angle β (C15···C10···C17···N4) of −20(1)°
fully comply with the β-turn arrangement. A more detailed
analysis of the ϕ and φ dihedral angles defines the presence of
the β-turn conformation of type-II′. The values of ϕi+1
=
51(1)°, φi+1 = −134(1)° and ϕi+2 = −105(1)°, φi+1 = 31(1)°
show a higher distortion with respect to the ideal values for the
second couple, that could be related to the geometrical
constrain of the five-membered ring (C10/C12/C13/N2/
C14). This latter assumes almost an envelope conformation as
described by the puckering parameters q2 = 0.216(4) Å, ϕ2 =
−153.2(8)°,24 with C12 forming the flap out of the best plane
of the remaining four atoms by 0.347(4) Å. The pyrrolidine is
nearly equatorially oriented with respect to the indole moiety
(dihedral angle between the least-squares planes of 87(1)°) and
the pendant residue linked to N2 is characterized by the torsion
angle C13−N2−C17−C19 of 74(1)°.
These X-ray results are noteworthy, as they demonstrate that
the intramolecular hydrogen bond persists also during the
crystal formation when the intermolecular packing forces are
crucial and, even more so, in the presence of a highly
coordinating protic solvent (MeOH). Unfortunately, we were
unable to obtain crystals suitable for an X-ray diffraction
analysis of mimic 19, under numerous protic solvent
conditions.
As a final remark, we found a significant geometrical
correspondence between the three-dimensional arrangement
of the experimental structure of 3 with the modeled
conformation, as provided by the rms value of 0.611 for the
superimposition of the whole molecule and by a value of 0.105
Å obtained by superimposition of the backbone amide atoms in
the β-turn region (see the Supporting Information).
However, since the intermolecular forces in the solid state
may lead to a change in the peptidomimetic secondary
structure, solution-phase studies for 3 and 19 were also
performed in order to give a complete representation of their
conformation and to assess the stability of the observed β-turn
in a biologically meaningful medium.
After full characterization by one- and two-dimensional
(COSY, HSQC) NMR analyses, an NMR study of conforma-
tional behavior was conducted. In similar constrained peptides,
intramolecular hydrogen bonding provides the principal driving
force for β-turn formation. In order to obtain validation about
Figure 5. ORTEP23 view of 3 showing a β-turned conformation and
the relative arbitrary atom-numbering scheme (thermal ellipsoids at
40% probability). H atoms are shown as spheres of arbitrary size.
synthetic approach, starting from a substrate with known
chirality (N-Boc-D-trp methyl ester), X-ray study first high-
lighted the stereochemistry of the two stereocenters, which are
both in the S configuration.
The molecule, with the exception of the pyrrolidine unit
linked via a spiro ring junction to the piperidine−indole system,
is almost flat due not only to the presence of fused aromatic
rings (angles between the calculated mean-square planes for the
three rings are in the range 1.0−5.3(1)°) but also to the
occurrence of a strong intramolecular hydrogen bond between
N4−H4 and O2 (ORTEP numbering, distance 2.08(1)Å, angle
172(1)°), closing a further 10-membered pseudocyclo
structure. This intramolecular hydrogen bond between the
COi oxygen atom acceptor and NHi+3 amide donor confers on
the crystal structure a β-turn conformation, characterized by an
almost ideal hydrogen bond directionality of the (i)O···H−N(i+3)
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the presence of such bonding, the conformational studies were
carried out in a relatively nonpolar solvent (chloroform), which
does not provide strong hydrogen-bonding competition. Such
solvent can be considered to mimic the hydrophobic core of a
protein; nevertheless, the possible influence of unpredictable
factors on the conformation in biological water should be not
excluded.
The involvement of the NH amide proton in intramolecular
hydrogen bonding was first estimated from evaluation of its
chemical shift value in CDCl3 and from variable-temperature
(VT) studies performed on a 2.0 mM CDCl3 solution of 3 and
19. The chemical shift of the NH amide proton ranged from
7.99 ppm (T = 238 K) to 7.80 ppm (T = 303 K) for compound
3 and from 7.82 ppm (T = 243 K) to 7.55 ppm (T = 323 K) for
compound 19. These values, in particular the two low VT
coefficients (Table 3), support the involvement of the amide
tion as somatostatin mimetics. Both computational studies and
spectroscopic NMR, IR, and X-ray investigations support a
stable type II′ β-turn conformation for compound 3,
embodying a constrained D-Trp-Ala dipeptide analogue as the
central i+1/i+2 core, with highly predictable stereostructural
properties. The turn properties of 3 are efficiently transferred to
the somatostatin mimic 19, in which the pharmacophore Lys
amino acid takes the place of Ala in the i+2 position. Also for
19 a type II′ β-turn conformation was predicted by computa-
tional calculations and strongly assessed with NMR experi-
ments.
Contrary to our expectations, biological evaluation of
trifluoroacetate salts 4 and 21 did not afford any acceptable
level of receptor affinity. Aimed to improve these results and
relying also on docking studies, a structure-based approach is
now underway, to evaluate possible structural modifications of
the described peptidomimetics or introduction of extra
recognition features for affinity.
1
Table 3. H NMR Data for the NHMe Proton in
a
Compounds 3 and 19
EXPERIMENTAL SECTION
■
δ
NHMe
compd (ppm)
Δδ/ΔT
(NHMe) (VT)
(ppb/K)
Δδ (NHMe)
(30% DMSO-d6)
(ppm)
NH/ND
(NHMe)
exchange (min)
Synthesis. (R)-Methyl 2-((tert-Butoxycarbonyl)amino)-3-(1-(trii-
sopropylsilyl)-1H-indol-3-yl)propanoate, 5. To a solution of (R)-
methyl 2-((tert-butoxycarbonyl)amino)-3-(1H-indol-3-yl)propanoate
(6.09 g, 19.13 mmol) in anhydrous THF (105 mL) cooled to −78
°C under nitrogen atmosphere was added dropwise KHMDS (1 M in
THF, 24.88 mL, 24.88 mmol). After 1 h, triisopropylsilyl chloride
(4.06 mL, 19.13 mmol) in THF (20 mL) was added dropwise at −78
°C, and the solution was allowed to warm to room temperature. After
the solution was quenched with H2O, THF was evaporated in vacuo,
and the aqueous solution was extracted with Et2O (3 × 30 mL). The
organic layer was washed with brine, dried over Na2SO4, and filtered,
and the solvent was removed under reduced pressure. Purification by
flash column chromatography (9:1, n-hexane/EtOAc) afforded 5 (8.04
3
7.69
7.59
−3.00
−3.00
0.13
0.19
210
300
19
a
NMR experiments were performed on a 2.0 mM solution in CDCl3.
proton in a hydrogen bond, in both molecules. Further,
titration of the CDCl3 2.0 mM solution with up to 30% of
DMSO-d6, a strongly coordinating and hydrogen bond-
acceptor solvent, produced a low variation of the NH chemical
shift from 7.69 to 7.82 ppm for 3 and from 7.59 to 7.78 ppm for
19,25 thus highlighting the high stability of the intramolecular
hydrogen bond present in both compounds.
Moreover, the rate of the H/D exchange upon addition of
CD3OD was quite low for both 3 and 19, once again
confirming the prevalence of an intramolecularly hydrogen-
bonded status for the NHMe proton.
g, 89%) as a foam: Rf 0.61 (7:3, n-hexane/EtOAc); [α]20 −9.5 (c
D
0.40, MeOH); 1H NMR (400 MHz, DMSO-d6) δ 7.51 (d, J = 7.6 Hz,
1H), 7.48 (d, J = 8.0 Hz, 1H), 7.24 (s, br, 1H), 7.21 (d, J = 8.1 Hz,
1H), 7.12 (t, J = 7.5 Hz, 1H), 7.06 (t, J = 7.4 Hz, 1H), 4.29−4.21 (m,
1H), 3.59 (s, 3H), 3.13 (dd, J = 14.5 and 5.2 Hz, 1H), 3.00 (dd, J =
14.5 and 9.5 Hz, 1H), 1.70 (ept, J = 7.4 Hz, 3H) 1.31 (s, 9H) 1.11−
1.055 (m, 18H); 13C NMR (100 MHz, DMSO-d6) δ 173.4, 155.8,
139.3, 136.6, 124.2, 121.4, 118.8, 118.4, 111.9, 110.2, 78.7, 55.1, 52.2,
28.6 (3C), 27.3, 18.3 (6C), 12.6 (3C); HRMS (EI) calcd for
C26H42N2O4Si 474.2914, found 474.2928.
The IR26 spectrum (2.0 mM solution CHCl3) of 3 exhibited
an extensive absorption band at 3355 cm−1 for the hydrogen-
bonded NH stretch and a minor band at 3467 cm−1 for the
non-hydrogen-bonded NH stretch, in accordance with the
presence of a predominant bonded state in this molecule. The
NH stretching region of the IR spectrum of 19 was
complicated, due to the presence of three different NH
functional groups, and did not provide useful indications about
the presence of intramolecular H-bonds.
Somatostatin regulates the endocrine system and affects
neurotransmission and cell proliferation via interaction with G-
protein-coupled receptors. Unexpectedly, Nε-deprotected
samples 4 and 21 did not inhibit the binding of 125I-
somatostatin-14 to the different human somatostatin receptors
(sst1-sst5), at concentrations up to 1 μM, that we retain
important for therapeutic activity. Somatostatin-28, tested in
parallel as reference compound, showed complete inhibition
with Ki values in the nM range, thus suggesting that further
interactions would be required for affinity, besides type II′ β-
turn recognition.
(R)-Methyl 2-Amino-3-(1-(triisopropylsilyl)-1H-indol-3-yl)-
propanoate, 6. To a solution of 5 (5.00 g, 10.53 mmol) in EtOAc
(200 mL) cooled to 0 °C was added SnCl4·H2O (4.94 mL, 42.13
mmol) was added. After 48 h at room temperature, the reaction
mixture was poured into satd aq NaHCO3 (100 mL), the layers were
separated, and the aqueous layer was extracted with EtOAc (3 × 50
mL). The combined organic layers were washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo. Purification by flash
chromatography (8:2, EtOAc/ n-hexane) afforded 6 (3.74 g, 95%
yield) as an oil: Rf 0.36 (8:2, EtOAc/n-hexane); [α]20D−24.5 (c 0.40,
1
MeOH); H NMR (400 MHz, DMSO-d6) 7.53 (d, br, J = 7.8 Hz,
1H), 7.47 (d, J = 8.1 Hz, 1H), 7.13 (s, 1H), 7.11 (m, 1H), 7.05 (m,
1H), 3.67 (m, 1H), 3.51 (s, 3H), 3.06−2.92 (m, 2H), 1.76 (d, J = 7.6
Hz, 2H), 1.70 (ept, J = 7.6 Hz, 3H), 1.08 (d, J = 7.6 Hz, 18H); 13C
NMR (100 MHz, DMSO-d6) δ 176.1, 141.1, 131.3, 130.0, 121.8,
119.7, 119.0, 114.1, 113.8, 55.4, 51.7, 31.2, 18.4 (6C), 12.5 (3C);
HRMS (EI) calcd for C21H34N2O2Si 374.2390, found 374.2385.
(R)-2-Amino-3-(1-(triisopropylsilyl)-1H-indol-3-yl)propanoic Acid,
7. To a solution of 6 (7.30 g, 19.49 mmol) in THF (195 mL) was
added aq LiOH 1 M (19.49 mL, 19.49 mmol). After 4 h at room
temperature, the solution was acidified to pH 3 with 5% aq H3PO4, the
THF was evaporated, and the aqueous solution was extracted with
EtOAc (3 × 30 mL). The combined organic extracts were washed with
brine, dried over Na2SO4, filtered, and concentrated in vacuo to afford
CONCLUSIONS
■
In summary, tetrahydro-β-carboline-based spirocyclic lactams
have been prepared with the purpose of exploring their
conformational behavior and evaluate their potential applica-
7 (6.36 g, 90% yield) as a foam: Rf 0.12 (8:2, EtOAc/MeOH); [α]20
D
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+18.3 (c 0.30, MeOH); 1H NMR (500 MHz, DMSO-d6) 7.59 (d, br, J
= 7.7 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.32 (s, 1H), 7.12 (m, 1H),
7.06 (t, br, J = 7.7 Hz, 1H), 3.73−2.90 (m, br, 3H), 3.45 (dd, J = 9.2
and 3.8 Hz, 1H), 3.33 (dd, J = 15.2 and 3.8 Hz, 1H), 2.95 (dd, J = 15.2
and 9.2 Hz, 1H), 1.71 (ept, J = 7.5 Hz, 3H), 1.10 (d, J = 7.5 Hz, 18H);
13C NMR (100 MHz, DMSO-d6) δ 170.4, 141.4, 131.2, 130.5, 121.8,
mixture was filtered, concentrated, and extracted with EtOAc (3 × 75
mL). The organic layer was washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo to afford a residue which was
dissolved in MeOH (90 mL). A solution of 7% aq NaIO4 (16 mL, 5.57
mmol) was added, and the solution was allowed to react for 24 h at
room temperature. After the addition of H2O (15 mL), the mixture
was extracted with EtOAc (3 × 50 mL), the organic layer was washed
with brine, dried over Na2SO4, and filtered, and the solvent was
removed under reduced pressure. Purification by flash chromatography
(98:2, n-hexane/EtOAc) afforded 10 (2.89 g, 90% yield) as a brown
oil: Rf 0.72 (7:3, n-hexane/EtOAc); [α]20D −38.2 (c 1.00, MeOH); 1H
NMR (400 MHz, DMSO-d6) δ 9.32 (s, br, 1H), 7.46 (d, J = 8.3 Hz,
1H), 7.43−7.31 (m, 6H), 7.10 (ddd, J = 8.3, 7.3, and 1.3 Hz, 1H), 7.06
(s, 1H), 6.99 (t, br, J = 7.3 Hz, 1H), 5.52 (s, 1H), 5.10 (d, J = 11.9 Hz,
1H), 5.03 (d, J = 11.9 Hz, 1H), 3.67 (d, br, J = 19.0 Hz, 1H), 3.33 (d, J
= 14.9 Hz, 1H), 3.27 (d, J = 14.9 Hz, 1H), 3.04 (d, J = 19.0 Hz, 1H),
1.65 (ept, J = 7.5 Hz, 3H), 1.09−1.03 (m, 18H), 0.63 (s, 9H); 13C
NMR (100 MHz, DMSO-d6) δ 200.7, 173.4, 154.8, 140.8, 135.8,
132.1, 131.9, 129.2 (2C), 129.0 (3C), 121.9, 120.0, 119.1, 114.1, 111.3,
95.6, 67.7, 63.9, 39.8, 37.3, 33.1, 25.9 (3C), 18.3 (6C), 12.5 (3C);
HRMS (EI) calcd for C35H48N2O5Si 604.3333, found 604.3346.
(S)-Methyl 2-((S)-3-(((Benzyloxy)carbonyl)amino)-2-oxo-3-((1-
(triisopropylsilyl)-1H-indol-3-yl)methyl)pyrrolidin-1-yl)propanoate,
11. To a solution of 10 (1.26 g, 2.08 mmol), NaOAc (0.36 g, 4.37
mmol), and activated powdered 4 Å molecular sieves (2.30 g) in
MeOH (45 mL) was added the hydrochloride salt of L-alanine methyl
ester (0.32 g, 2.29 mmol). After 1 h, NaCNBH3 (0.26 g, 4.17 mmol)
was added, and the mixture was stirred at room temperature overnight.
Then the suspension was filtered, and the filtrate was acidified to pH 1
with aq HCl 1 N and stirred for 15 min. The solution was basified to
pH 9 with saturated aq NaHCO3 and then extracted with EtOAc (3 ×
50 mL). The combined organic layers were washed with brine, dried
over Na2SO4, filtered, and concentrated in vacuo to give a colorless oil.
This oil was dissolved in toluene (25 mL), and HOBt monohydrate
(0.32 g, 2.08 mmol) was added. The mixture was heated at reflux for 6
h, and then it was cooled and saturated aq NaHCO3 (20 mL) added.
The mixture was extracted with EtOAc (3 × 30 mL), and the
combined organic layers were washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo. Purification by flash chromatog-
raphy (3:1, n-hexane/EtOAc) afforded 11 (0.890 g, 70% yield) as a
119.6, 119.0, 114.1, 113.7, 54.8, 27.7, 18.5 (6C), 12.6 (3C); HRMS
(EI) calcd for C20H32N2O2Si 360.2233, found 360.2250.
(2R,4R)-Benzyl 2-tert-Butyl-5-oxo-4-((1-(triisopropylsilyl)-1H-
indol-3-yl)methyl)oxazolidine-3-carboxylate, 8. To a solution of 7
(6.17 g, 17.11 mmol) in EtOH (55 mL) was added aq NaOH 1 M
(17.11 mL, 17.11 mmol). After 1 h under stirring at room
temperature, the solution was concentrated in vacuo to provide a
white solid. The solid was suspended in anhydrous CH2Cl2 (125 mL),
and pivalaldehyde (2.79 mL, 25.67 mmol) was added under nitrogen
atmosphere. The flask was equipped with a Soxhlet filled with
molecular sieves (3 Å), and the solution was heated to reflux for 12 h.
After cooling, the mixture was concentrated in vacuo to provide a
yellow foam that was azeotropically dried with toluene (35 mL). The
resultant solid residue was dissolved in anhydrous CH2Cl2 (125 mL),
and benzyl chloroformate (3.18 mL, 22.25 mmol) was added at 0 °C
under nitrogen atmosphere. After 48 h at 4 °C, the solution was
allowed to warm to room temperature. Water (20 mL) and DMAP (6
mg) were added, and the solution was stirred for 1 h; then the layers
were separated and the aqueous layer was extracted with CH2Cl2 (3 ×
20 mL). The combined organic layers were washed with 15% aq
NaHSO3 and saturated aq NaHCO3, dried over Na2SO4, filtered, and
concentrated in vacuo. Purification by flash chromatography (95:5, n-
hexane/EtOAc) afforded 8 (7.39 g, 77% yield) as an oil: Rf 0.61 (7:3,
n-hexane/EtOAc); [α]20D −8.6 (c 1.25, MeOH); 1H NMR (400 MHz,
DMSO-d6) 7.45 (d, J = 8.3 Hz, 1H), 7.43−7.32 (m, 6H), 7.15 (s, 1H),
7.07 (t, br, J = 7.8 Hz, 1H), 6.89 (t, br, J = 7.6 Hz, 1H), 5.45 (s, 1H),
5.14 (d, J = 12.4 Hz, 1H), 5.05 (d, J = 12.4 Hz, 1H), 4.64 (dd, J = 8.3
and 5.1 Hz, 1H), 3.30−3.17 (m, 2H), 1.67 (ept, J = 7.5 Hz, 3H) 1.06
(d, J = 7.5 Hz, 18H), 0.81 (s, 9H); 13C NMR (125 MHz, DMSO-d6) δ
172.1, 156.4, 141.1, 136.3, 131.2, 131.1, 128.9 (3C), 128.6 (2C), 121.8,
119.7, 119.0, 114.1, 112.7, 95.6, 67.7, 57.8, 36.9, 28.4, 25.2 (3C), 18.3
(6C), 12.5 (3C); HRMS (EI) calcd for C33H46N2O4Si 562.3227,
found 562.3215.
(2R,4R)-Benzyl 4-Allyl-2-tert-butyl-5-oxo-4-((1-(triisopropylsilyl)-
1H-indol-3-yl)methyl)oxazolidine-3-carboxylate, 9. To a solution
of 8 (7.30 g, 12.97 mmol) in anhydrous THF (150 mL) was added
KHMDS (1 M in toluene, 18.16 mL, 18.16 mmol) dropwise at −78
°C under nitrogen atmosphere. After 1 h, allyl bromide (1.23 mL,
14.27 mmol) was added dropwise at −78 °C, and the solution was
allowed to warm to −30 °C before quenching with saturated aq
NH4Cl (30 mL). At 0 °C the mixture was diluted with water (30 mL)
and extracted with EtOAc (3 × 50 mL). The organic layer was washed
with brine, dried over Na2SO4, and filtered, and the solvent was
removed under reduced pressure. Purification by flash chromatography
(98:2, n-hexane/EtOAc) afforded 9 (6.40 g, 82% yield) as an oil: Rf
0.49 (8:2, n-hexane/EtOAc); [α]20D −65.5 (c 0.45, MeOH); 1H NMR
(400 MHz, DMSO-d6) δ 7.65−7.29 (m, 7H), 7.05 (t, J = 7.4 Hz, 1H),
7.03−6.87 (m, 2H), 5.52 (dddd, J = 18.7, 10.1, 8.6, and 6.3 Hz, 1H),
5.40−5.30 (m, br, 1H), 5.28 (s, 1H), 5.28−5.19 (m, br, 1H), 5.15 (d,
br, J = 10.1 Hz, 1H), 5.02 (dd, J = 18.7 and 1.5 Hz, 1H), 3.55−3.37
(m, br, 1H), 3.17−2.99 (m, br, 1H), 2.52−2.37 (m, 2H), 1.60 (ept, br,
J = 7.5 Hz, 3H), 1.04 (d, J = 7.5 Hz, 18H), 0.31 (s, 9H); 13C NMR
(100 MHz, DMSO-d6) δ 173.8, 155.9, 140.8, 135.9, 132.1, 131.9,
130.9, 129.6 (2C), 129.1 (3C), 122.1, 121.8, 119.8, 119.2, 114.0, 112.2,
95.0, 69.0, 67.9, 39.5, 37.6, 32.1, 25.2 (3C), 18.3 (6C),12.5 (3C);
HRMS (EI) calcd for C36H50N2O4Si 602.3540, found 602.3529.
(2R,4S)-Benzyl 2-tert-Butyl-5-oxo-4-(2-oxoethyl)-4-((1-(triisopro-
pylsilyl)-1H-indol-3-yl)methyl)oxazolidine-3-carboxylate, 10. To a
solution of NMO (50 wt % solution in H2O, 3.30 mL, 15.92 mmol) in
acetone/H2O 6:1 (49 mL), OsO4 (2 wt % solution in acetone/H2O
1:1, 3.73 mL, 0.26 mmol) was added; the solution of 9 (3.20 g, 5.31
mmol) in acetone (90 mL) was then added, and the mixture was
allowed to react for 20 h under nitrogen atmosphere. After the
addition of 10% aq NaHSO3 (45 mL) and 1 h under stirring, the
yellow oil: Rf 0.54 (3:1, n-hexane/EtOAc); [α]20 −36.7 (c 0.95,
D
MeOH); 1H NMR (400 MHz, DMSO-d6) δ 7.52 (m, br, 1H), 7.48 (d,
br, J = 7.5 Hz, 1H), 7.46 (d, br, J = 7.6 Hz, 1H), 7.44−7.29 (m, 5H),
7.09 (ddd, J = 8.1, 7.1, and 1.3 Hz, 1H), 7.01 (t, br, J = 8.1 Hz, 1H),
5.02 (m, br, 2H), 4.25 (q, J = 7.3 Hz, 1H), 3.55 (s, br, 3H), 3.13 (d, J =
14.2 Hz, 1H), 3.08−2.98 (m, 2H), 2.47−2.27 (m, 2H), 2.20−2.09 (m,
1H), 1.74 (ept, J = 7.6 Hz, 3H), 1.09 (d, J = 7.6 Hz, 9H), 1.07 (d, J =
7.6 Hz, 9H), 0.19 (d, J = 7.3 Hz, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 173.6, 171.8, 155.0, 140.8, 137.6, 136.3, 128.8(3C), 128.2(2C),
125.4, 121.4, 118.9, 118.6, 111.8, 107.8, 65.6, 61.6, 52.3, 49.6, 39.6,
32.7, 28.8, 18.3 (6C), 13.6, 12.6 (3C); HRMS (EI) calcd for
C34H47N3O5Si 605.3285, found 605.3277.
(S)-Methyl 2-((S)-3-((1H-Indol-3-yl)methyl)-3-amino-2-oxopyrroli-
din-1-yl)propanoate, 12. To a solution of 11 (0.850 g, 1.40 mmol) in
MeOH (30 mL) was added Pd/C 10% (85 mg). After thoroughly
flushing the flask with N2, a hydrogen atmosphere was introduced.
After 17 h, the reaction was filtered and the solvent was evaporated in
vacuo to afford a yellow oil: Rf 0.43 (5:1, EtOAc/MeOH). The oil was
dissolved in THF (15 mL), and TBAF (1 M in THF, 2.8 mL, 2.8
mmol) was added at 0 °C. After 1 h under stirring, the mixture was
diluted with EtOAc (30 mL), washed with saturated aq NaHCO3 and
brine, dried over Na2SO4, filtered, and concentrated in vacuo.
Purification by flash chromatography (85:15, EtOAc/MeOH) afforded
12 (0.360 g, 82% yield) as a yellow oil: Rf 0.27 (85:15, EtOAc/
MeOH); [α]20D−62.3 (c 0.33, MeOH); 1H NMR (400 MHz, DMSO-
d6) δ 10.88 (m, br, 1H), 7.53 (d, J = 7.9 Hz, 1H), 7.32 (d, J = 8.1 Hz,
1H), 7.17 (d, J = 2.3 Hz, 1H), 7.04 (ddd, J = 8.1, 6.8, and 1.0 Hz, 1H),
6.95 (ddd, J = 8.1, 7.1, and 1.0 Hz, 1H), 4.45 (q, J = 7.3 Hz, 1H), 3.60
(s, 3H), 3.03 (td, J = 9.1 and 3.0 Hz, 1H), 2.94 (d, J = 13.9 Hz, 1H),
2.79 (d, J = 13.9 Hz, 1H), 2.47 (dt, J = 9.1 and 7.8 Hz, 1H), 2.12 (ddd,
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J = 12.6, 7.8, and 3.0 Hz, 1H), 1.88 (m, br, 2H), 1.74 (ddd, J = 12.6,
9.1, and 7.8 Hz, 1H), 0.78 (d, J = 7.3 Hz, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 177.8, 172.0, 136.3, 128.4, 124.8, 121.2, 118.9, 118.7,
111.7, 109.7, 60.4, 52.5, 49.6, 40.1, 34.9, 32.1, 14.0; HRMS (EI) calcd
for C17H21N3O3 315.1583, found 315.1565.
was added, and the mixture was stirred at room temperature overnight.
Then the suspension was filtered, and the filtrate was acidified to pH 4
with aq HCl 1 N and stirred for 15 min. The solution was basified to
pH 9 with saturated aq NaHCO3 and then extracted with EtOAc (3 ×
20 mL). The combined organic layers were washed with brine, dried
over Na2SO4, filtered, and concentrated in vacuo to give a yellow oil.
The oil was dissolved in toluene (15 mL), and HOBt monohydrate
(0.089 g, 0.58 mmol) was added. The mixture was heated at reflux for
6 h, and then it was cooled and saturated aq NaHCO3 (10 mL) added.
The mixture was extracted with EtOAc (3 × 20 mL), and the
combined organic layers were washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo. Purification by flash chromatog-
raphy (8:2, n-hexane/EtOAc) afforded 15 (0.312 g, 71% yield) as a
(S)-Methyl2-((S)-2′-Oxo-1,2,4,9-tetrahydrospiro[pyrido[3,4-b]-
indole-3,3′-pyrrolidin]-1′-yl)propanoate, 13. To a solution of 12
(0.435 g, 1.38 mmol) in MeOH (45 mL) were added activated
powdered 4 Å molecular sieves (1.30 g), formaldehyde (37 wt %
solution in H2O, 0.123 mL, 1.66 mmol) and trifluoroacetic acid (0.11
mL, 1.38 mmol). After 7 h under stirring, molecular sieves were
filtered, and the mixture was basified with saturated aq NaHCO3 and
extracted with CH2Cl2 (3 × 10 mL). The combined organic layers
were dried over Na2SO4, filtered, and concentrated in vacuo.
Purification by flash chromatography (95:5, EtOAc/MeOH) afforded
13 (0.298 g, 66% yield) as a foam: Rf 0.43 (4:1, EtOAc/MeOH);
[α]20D +9.5 (c 0.33, MeOH); 1H NMR (400 MHz, DMSO-d6) δ 10.69
(s, br, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.27 (d, J = 8.1 Hz, 1H), 7.01
(ddd, J = 8.1, 7.1, and 1.3 Hz, 1H), 6.93 (ddd, J = 7.8, 7.1, and 1.0 Hz,
1H), 4.66 (q, J = 7.3 Hz, 1H), 4.00 (d, br, J = 16.8 Hz, 1H), 3.88 (d,
br, J = 16.8 Hz, 1H), 3.68 (s, 3H), 3.41−3.35 (m, 2H), 2.71−2.59 (m,
2H), 2.52−2.34 (m, br, 1H), 2.02 (dt, J = 12.4 and 7.9 Hz, 1H), 1.85
(ddd, J = 12.4, 6.9, and 5.1 Hz, 1H), 1.39 (d, J = 7.3 Hz, 3H); 13C
NMR (100 MHz, DMSO-d6) δ 175.9, 171.9, 136.3, 133.5, 127.9,
120.8, 118.6, 117.6, 111.3, 105.3, 59.1, 52.6, 49.8, 41.1, 39.2, 30.7, 28.2,
15.0; HRMS (EI) calcd for C18H21N3O3 327.1583, found 327.1597.
(S)-N-Methyl-2-((S)-2′-oxo-1,2,4,9-tetrahydrospiro[pyrido[3,4-b]-
indole-3,3′-pyrrolidin]-1′-yl)propanamide, 14. To a solution of 13
(0.305 g 0.93 mmol) in THF (20 mL) was added methanamine (2 M
in THF, 3.73 mL, 7.45 mmol). After 6 h under stirring, the mixture
was concentrated in vacuo to afford pure 14 (0.300 g, 98% yield) as a
yellow oil: Rf 0.23 (6:4, n-hexane/EtOAc); [α]20 −23.9 (c 1.00,
D
MeOH); 1H NMR (400 MHz, DMSO-d6) δ 7.52−7.44 (m, 3H), 7.42
(s, 1H), 7.40−7.29 (m, 5H), 7.10 (t, br, J = 7.7 Hz, 1H), 7.00 (t, J =
7.5 Hz, 1H), 6.68 (m, br, 1H), 5.01 (m, br, 2H), 4.12 (dd, J = 9.3 and
6.1 Hz, 1H), 3.56 (s, br, 3H), 3.19−3.08 (m, 2H), 3.00 (d, br, J = 13.6
Hz, 1H), 2.80−2.66 (m, 2H), 2.41−2.23 (m, 3H), 1.74 (ept, J = 7.4
Hz, 3H), 1.38 (s, 9H), 1.09 (d, J = 7.4 Hz, 9H), 1.08 (d, J = 7.4 Hz,
9H), 1.15−0.88 (m, br, 3H), 0.88−0.61 (m, br, 2H), 0.02 (m, br, 1H);
13C NMR (100 MHz, DMSO-d6) δ 173.7, 171.2. 170.1, 155.0, 140.8,
137.6, 136.5, 128.8 (3C), 128.2(2C), 128.0, 122.0, 119.6, 119.0, 114.1,
111.2, 77.7, 66.6, 65.6, 54.5, 52.2, 39.9(2 C), 32.3, 29.3, 28.7 (3C),
28.6, 28.0, 23.0, 18.3 (6C), 12.4 (3C); HRMS (EI) calcd for
C42H62N4O7Si 762.4388, found 762.4392.
(S)-Methyl 2-((S)-3-((1H-Indol-3-yl)methyl)-3-amino-2-oxopyrroli-
din-1-yl)-6-((tert-butoxycarbonyl)amino)hexanoate, 16. To a sol-
ution of 15 (0.300 g, 0.39 mmol) in MeOH (14 mL) was added Pd/C
10% (30 mg). After thoroughly the flask was thoroughly with N2, a
hydrogen atmosphere was introduced. After 12 h, the reaction was
filtered, and the solvent was evaporated in vacuo to afford a yellow oil:
Rf 0.45 (4:1, EtOAc/MeOH). The oil was dissolved in THF (6 mL),
and TBAF (1 M in THF, 0.786 mL, 0. 786 mmol) was added at 0 °C.
After 1 h under stirring, the mixture was diluted with EtOAc (20 mL),
washed with saturated aq NaHCO3 and brine, dried over Na2SO4,
filtered, and concentrated in vacuo. Purification by flash chromatog-
raphy (85:15, EtOAc/MeOH) afforded 16 (0.167 g, 90% yield) as a
yellow foam: Rf 0.31 (3:1, EtOAc/MeOH); [α]20 −7.5 (c 0.2
D
MeOH); 1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, br, 1H), 7.82 (q,
br, J = 4.5 Hz, 1H), 7.35 (d, br, J = 7.8 Hz, 1H), 7.27 (d, J = 7.8 Hz,
1H), 7.01 (ddd, J = 7.8, 7.3, and 1.3 Hz, 1H), 6.93 (ddd, J = 7.8, 7.3,
and 1.0 Hz, 1H), 4.53 (q, J = 7.3 Hz, 1H), 4.00 (d, J = 16.9 Hz, 1H),
3.88 (d, br, J = 16.9 Hz, 1H), 3.49 (td, J = 9.3 and 3.5 Hz, 1H), 3.40−
3.25 (m, br, 1H), 3.36 (dt, J = 9.3 and 7.3 Hz, 1H), 2.68 (d, br, J =
15.2 Hz, 1H), 2.62 (d, J = 15.2 Hz, 1H), 2.61 (d, J = 4.5 Hz, 3H), 2.03
(ddd, J = 12.2, 9.3, and 7.3 Hz, 1H), 1.81 (ddd, J = 12.2, 7.3, and 3.5
Hz, 1H), 1.30 (d, J = 7.3 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ
175.9, 171.3, 136.3, 133.5, 127.9, 120.8, 118.6, 117.6, 111.3, 105.4,
59.3, 50.2, 40.8, 39.3, 30.5, 28.1, 26.07, 15.5; HRMS (EI) calcd for
C18H22N4O2 326.1743, found 326.1736.
foam: Rf 0.32 (8:2, EtOAc/MeOH); [α]20 −61.4 (c 0.50, MeOH);
D
1H NMR (400 MHz, DMSO-d6) δ 10.86 (s, br, 1H), 7.54 (d, J = 7.8
Hz, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.15 (d, J = 2.3 Hz, 1H), 7.04 (ddd,
J = 7.8, 7.1, and 1.0 Hz, 1H), 6.96 (ddd, J = 7.8, 7.1, and 1.0 Hz, 1H),
6.72 (m, br, 1H), 4.37 (dd, J = 9.3, 6.1 Hz, 1H), 3.62 (s, 3H), 3.17 (td,
J = 8.9 and 3.8 Hz, 1H), 2.98−2.68 (m, 5H), 2.08 (ddd, J = 12.4, 7.6,
and 3.8 Hz, 1H), 2.03−1.88 (m, 2H), 1.71 (ddd, J = 15.7, 8.6, and 7.1
Hz, 1H), 1.62−1.48 (m, 1H), 1.37 (s, 9H), 1.29−1.19 (m, 3H), 1.01−
0.79 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 178.0, 171.6, 156.1,
136.3, 128.5, 124.9, 121.2, 118.9, 118.8, 111.7, 109.7, 77.9, 60.6, 54.0,
52.4, 39.9 (2C), 34.3, 31.9, 29.3, 28.7 (3C), 28.1, 23.2; HRMS (EI)
calcd for C25H36N4O5 472.2686, found 472.2693.
(S)-Methyl 6-((tert-Butoxycarbonyl)amino)-2-((S)-2′-oxo-1,2,4,9-
tetrahydrospiro[pyrido[3,4-b]indole-3,3′-pyrrolidin]-1′-yl)-
hexanoate, 17. To a solution of 16 (0.100 g, 0.21 mmol) in MeOH
(7 mL) were added activated powdered 4 Å molecular sieves (0.200
g), formaldehyde (37 wt % solution in H2O, 0.019 mL, 0.25 mmol),
and trifluoroacetic acid (0.016 mL, 0.21 mmol). After 5 h under
stirring, molecular sieves were filtered, and the mixture was basified
with saturated aq NaHCO3 and extracted with CH2Cl2 (3 × 10 mL).
The combined organic layers were dried over Na2SO4, filtered, and
concentrated in vacuo. Purification by flash chromatography (98:2,
(S)-2-((S)-2-Acetyl-2′-oxo-1,2,4,9-tetrahydrospiro[pyrido[3,4-b]-
indole-3,3′-pyrrolidin]-1′-yl)-N-methylpropanamide, 3. To a sol-
ution of 14 (0.285 g, 0.87 mmol) in CH2Cl2 (13 mL) were added
DIPEA (0.229 mL, 1.31 mmol) and acetic anhydride (0.124 mL, 1.31
mmol). After 16 h under stirring, the mixture was concentrated in
vacuo to give a yellow foam. Crystallization (5% MeOH in EtOAc)
afforded 3 (0.233 g, 72% yield) as a white solid: Rf 0.59 (3:1, EtOAc/
1
MeOH); [α]20D−63.2 (c 0.10, MeOH); mp 287−289 °C; H NMR
(400 MHz, DMSO-d6) δ 11.00 (s, br, 1H), 7.87 (q, br, J = 4.5 Hz,
1H), 7.49 (d, br, J = 7.8 Hz, 1H), 7.37 (d, br, J = 8.1 Hz, 1H), 7.09
(ddd, J = 8.1, 7.2, and 1.3 Hz, 1H), 7.00 (ddd, J = 7.8, 7.2, and 1.0 Hz,
1H), 4.91 (dd, br, J = 15.5 and 1.2 Hz, 1H), 4.73 (d, J = 15.5 Hz, 1H),
4.53 (q, J = 7.4 Hz, 1H), 3.40 (q, br, J = 8.8 Hz, 1H), 3.20 (td, J = 9.8
and 2.5 Hz, 1H), 2.99 (d, J = 14.8 Hz, 1H), 2.85 (d, br, J = 14.8 Hz,
1H), 2.67 (d, J = 4.5 Hz, 3H), 2.22 (s, 3H), 2.09 (m, 1H), 1.79 (ddd, J
= 12.3, 8.8, and 2.5 Hz, 1H), 1.34 (d, J = 7.4 Hz, 3H); 13C NMR (125
MHz, DMSO-d6) δ 173.7, 172.5, 171.0, 136.8, 130.6, 126.9, 121.5,
119.1, 118.2, 111.7, 104.3, 62.8, 50.3, 43.2, 39.8, 30.4, 29.4, 26.4, 23.6,
14.1; HRMS (EI) calcd for C20H24N4O3 368.1848, found 368.1843.
(S)-Methyl 2-((S)-3-(((Benzyloxy)carbonyl)amino)-2-oxo-3-((1-
(triisopropylsilyl)-1H-indol-3-yl)methyl)pyrrolidin-1-yl)-6-((tert-
butoxycarbonyl)amino)hexanoate, 15. To a solution of 10 (0.350 g,
0.58 mmol), NaOAc (0.099 g, 1.22 mmol), and activated powdered 4
Å molecular sieves (0.65 g) in MeOH (18 mL) was added (S)-methyl
2-amino-6-((tert-butoxycarbonyl)amino)hexanoate hydrochloride
(0.189 g, 0.64 mmol). After 1 h, NaCNBH3 (0.072 g, 1.16 mmol)
EtOAc/n-hexane) afforded 17 (72 mg, 70% yield) as a foam: Rf 0.53
1
(7:3, EtOAc/MeOH); [α]20 + 9.5 (c 0.33, MeOH); H NMR (400
D
MHz, DMSO-d6) δ 10.69 (s, br, 1H), 7.38 (d, br, J = 7.8 Hz, 1H), 7.27
(d, br, J = 8.1 Hz, 1H), 7.01 (t, br, J = 7.5 Hz, 1H), 6.92 (t, br, J = 7.4
Hz, 1H), 6.79 (m, br, 1H), 4.58 (dd, J = 10.5 and 5.2 Hz, 1H), 4.00 (d,
J = 16.7 Hz, 1H), 3.88 (d, br, J = 16.7 Hz, 1H), 3.68 (s, 3H), 3.40 (td, J
= 9.0 and 3.3 Hz, 1H), 3.36−3.26 (m, 1H), 2.95 ( (m, 2H), 2.70 (d, J
= 15.2 Hz, 1H), 2.63 (d, J = 15.2 Hz, 1H), 2.42 (m, br, 1H), 2.03 (dt, J
= 12.4 and 7.8 Hz, 1H), 1.91−1.71 (m, 3H), 1.49−1.32 (m, 2H), 1.37
(s, 9H), 1.32−1.19 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ
2607
dx.doi.org/10.1021/jo302737j | J. Org. Chem. 2013, 78, 2600−2610

The Journal of Organic Chemistry
Article
1
176.4, 171.5, 156.1, 136.3, 133.5, 127.9, 120.8, 118.6, 117.7, 111.3,
105.3, 77.8, 60.2, 59.2, 53.9, 52.5, 40.9, 39.2, 30.7, 29.3, 28.7 (3C),
28.4, 28.3, 23.2; HRMS (EI) calcd for C26H36N4O5 484.2686, found
484.2675.
0.46 (EtOAc); [α]20 +31.8 (c 0.50, MeOH); H NMR (400 MHz
D
CDCl3) δ 8.94 (m, br, 0.8H), 8.84 (m, br, 0.2H), 7.74−7.63 (m, 1H),
7.61−7.49 (m 1.5H), 7.40−7.09 (m, 13.5H), 6.76 (d, br, J = 6.1 Hz,
0.8H), 6.67 (d, br, J = 6.7 Hz, 0.2H), 5.26 (dt, J = 6.2 and 8.8 Hz,
0.4H), 5.17−5.03 (m, 1.2H), 4.88−4.59 (m, 3H), 4.50 (d, br, J = 14.6
Hz, 0.4H), 4.28 (d, br, J = 14.6 Hz, 0.4H), 4.18 (d, br, J = 14.3 Hz,
0.6H), 3.32−2.93 (m, 9H), 2.8−2.79 (m, 4H), 2.31 (m, 1H), 2.03 (m,
1H), 1.96 (s, 0.1H), 1.94 (s, 1H), 1.93 (s, 1.9H), 1.89−1.80 (m, 1H),
1.74−1.21 (m, 5H), 1.45 (s, 9H); 13C NMR (100 MHz CDCl3) δ
174.4, 173.0, 172.7, 172.6, 171.4, 156.8, 140.4 (2C), 136.1, 136.0,
129.8 (4C), 128.6 (4C), 127.2, 127.1, 126.6, 123.6, 120.0, 118.3, 111.0,
105.8, 83.2, 66.9, 55.3, 51.7 (2C), 42.4, 41.8, 40.5, 39.2, 39.1, 30.4, 29.6
(2C), 28.5 (3C), 28.1, 26.6, 26.4, 24.0; HRMS (EI) calcd for
C46H57N7O7 819.4319, found 819.4342.
To a solution of 20 (20 mg 0.024 mmol) in CH2Cl2 (1 mL) was
added 20% TFA in CH2Cl2 (1 mL). After 1 h under stirring, the
mixture was concentrated in vacuo to afford 21 (20 mg, 99% yield) as
a hygroscopic solid: HRMS (EI) calcd for C41H50N7O5 720.3868,
found 720.3884. Anal. Calcd for C43H50F3N7O7: C, 61.93; H, 6.04; N,
11.76. Found: C, 61.99; H, 6.05; N, 11.66.
tert-Butyl ((S)-6-(Methylamino)-6-oxo-5-((S)-2′-oxo-1,2,4,9-
tetrahydrospiro[pyrido[3,4-b]indole-3,3′-pyrrolidin]-1′-yl)hexyl)-
carbamate, 18. To a solution of 17 (0.210 g, 0.43 mmol) in EtOH
(0.5 mL), methanamine (8 M in EtOH, 3 mL, 24 mmol) was added.
After 5 h under stirring, the mixture was concentrated in vacuo to
afford pure 18 (0.207 g, 99% yield) as a foam: Rf 0.32 (7:3, EtOAc/
MeOH); [α]20D +15.9 (c 0.57 MeOH); 1H NMR (400 MHz, DMSO-
d6) δ 10.71 (s, br, 1H), 7.93 (m, br, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.28
(d, J = 7.8 Hz 1H), 7.01 (t, br, J = 7.5 Hz, 1H), 6.93 (t, br, J = 7.5 Hz,
1H), 6.78 (m, br, 1H), 4.45 (dd, J = 10.1 and 5.8 Hz, 1H), 4.43 (m, br,
1H), 4.02 (d, J = 16.8 Hz, 1H), 3.90 (d, J = 16.8 Hz, 1H), 3.59 (td, J =
9.1 and 3.2 Hz, 1H), 3.37−3.25 (m, 1H), 2.99−2.89 (m, 2H), 2.71 (d,
J = 14.9 Hz, 1H), 2.64 (d, J = 14.9 Hz, 1H), 2.60 (d, J = 4.5 Hz, 3H),
2.02 (dt, J = 12.4 and 7.9 Hz, 1H), 1.86−1.58 (m, 3H), 1.47−1.34 (m,
2H), 1.37 (s, 9H), 1.31−1.08 (m, 2H); 13C NMR (100 MHz CDCl3)
δ 176.7, 170.1, 156.1, 136.1, 131.8, 127.6, 121.8, 119.5, 117.8, 110.9,
106.2, 79.2, 59.9, 55.2, 40.9, 40.3, 39.2, 29.7, 29.5, 28.5 (3C), 28.0,
27.5, 26.3, 23.3; HRMS (EI) calcd for C26H37N5O4 483.2846, found
483.2861.
COMPUTATIONAL DETAILS
■
Conformational analysis was performed with the software Spartan
’0827 by means of the “conformer distribution” function using the
default search method (“Systematic” or “Monte Carlo” is automatically
chosen as that which leads to the smaller number of moves, depending
on the number of rotatable bonds in the molecule). The MMFF force
field was used for the energy minimization of the found structures. The
structures were then clustered according to the default setting of the
software (which consists in pruning out higher energy conformers and
keeping a diverse set of the low energy conformers using the RMS
torsion definition of nearness). Superimposition of the global
minimum of compounds 3 and 4 with standard β-turn types was
made with the alignment tool of the software and measured with the
alignment score function. Images were generated with the software
pyMol.28
(S)-5-((S)-2-((S)-2-Acetamido-3-phenylpropanoyl)-2′-oxo-1,2,4,9-
tetrahydrospiro[pyrido[3,4-b]indole-3,3′-pyrrolidin]-1′-yl)-6-(meth-
ylamino)-6-oxohexan-1-aminium 2,2,2-trifluoroacetate, 4. To a
solution of (S)-2-acetamido-3-phenylpropanoic acid (50 mg, 0.24
mmol) in CH2Cl2 (2 mL) were added DIPEA (0.084 mL, 0.48 mmol)
and HATU (92 mg, 0.24 mmol) under nitrogen atmosphere. After 1 h
under stirring, a solution of 18 (0.175 g, 0.36 mmol) and DIPEA
(0.084 mL, 0.48 mmol) in CH2Cl2 (2 mL) was added, and the mixture
was stirred at room temperature for 48 h. The solution was washed
with 5% aq H3PO4 and with saturated aq NaHCO3, dried over
Na2SO4, filtered, and concentrated in vacuo. Purification by flash
chromatography (98:2, EtOAc/MeOH) afforded 19 (71 mg, 44%
yield) as a foam: Rf 0.54 (8:2, EtOAc/MeOH); [α]20 +22.9 (c 0.72,
D
MeOH); 1H NMR (400 MHz CDCl3) δ 8.59 (m, br, 1H), 7.61 (m, br,
1H), 7.55 (d, J = 7.9 Hz, 1H), 7.42 (d, J = 7.3 Hz, 2H), 7.34 (d, J = 8.1
Hz, 1H), 7.33−7.26 (m, 2H), 7.23−7.18 (m, 2H), 7.15 (t, J = 7.6 Hz,
1H), 6.21 (m, br, 1H), 5.19 (dt, J = 7.0 and 7.3 Hz, 1H), 4.97 (d, br, J
= 14.4 Hz, 1H), 4.73 (d, br, J = 9.7 Hz, 1H), 4.66 (m, br, 1H), 4.37 (d,
br, J = 14.4 Hz, 1H), 3.27−3.08 (m, 6H), 3.03 (dd, J = 13.7 and 6.7
Hz, 1H), 2.86 (d, J = 4.7 Hz, 3H), 2.92−2.79 (m, 1H), 2.40−2.24 (m,
1H), 2.03 (s, 3H), 2.00−1.30 (m, 7H), 1.46 (s, 9H); 13C NMR (100
MHz CDCl3) δ 175.4, 172.3 172.0, 170.9, 156.7, 140.8, 136.4 135.8,
129.7 (2C), 128.5 (2C), 127.1, 126.5, 122.4, 120.0, 118.1, 111.2, 105.8,
80.9, 66.9, 55.0, 51.7, 42.7, 40.5, 39.8, 39.2, 30.4, 29.7, 29.6, 28.5 (3C),
26.7, 26.6, 23.6, 23.1; HRMS (EI) calcd for C37H48N6O6 672.3635,
found 672.3618.
ASSOCIATED CONTENT
* Supporting Information
■
S
General procedures and NMR spectra for all compounds; VT
NMR studies, DMSO-d6 titrations, and IR data for compounds
3 and 19; crystallographic data for compound 3 (CIF);
computational details. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: alessandra.silvani@unimi.it, manumuso@libero.it.
To a solution of 19 (35 mg 0.052 mmol) in CH2Cl2 (1 mL) was
added 20% TFA in CH2Cl2 (1 mL). After 1 h under stirring at room
temperature, the mixture was concentrated in vacuo to afford 4 (35
mg, 98% yield) as a hygroscopic solid: HRMS (EI) calcd for
C32H41N6O4 573.3184, found 573.3167. Anal. calcd for
C34H41F3N6O6: C, 59.47; H, 6.02; N, 12.24. Found: C 59,58; H,
6.10; N, 12.31.
(S)-5-((S)-2-((S)-2-((S)-2-Acetamido-3-phenylpropanamido)-3-
phenylpropanoyl)-2′-oxo-1,2,4,9-tetrahydrospiro[pyrido[3,4-b]-
indole-3,3′-pyrrolidin]-1′-yl)-6-(methylamino)-6-oxohexan-1-ami-
nium 2,2,2-Trifluoroacetate 21. To a solution of (S)-2-((S)-2-
acetamido-3-phenylpropanamido)-3-phenylpropanoic acid (44 mg,
0.12 mmol) in CH2Cl2 (2 mL) were added DIPEA (0.043 mL, 0.24
mmol), HATU (47 mg, 0.12 mmol), and HOAt (0.6 M in DMF, 0.207
mL, 0.12 mmol) under nitrogen atmosphere. After 1 h under stirring, a
solution of 18 (90 mg, 0.19 mmol) and DIPEA (0.043 mL, 0.24
mmol) in CH2Cl2 (2 mL) was added. The mixture was stirred at room
temperature for 48 h. The reaction mixture was washed with 5% aq
H3PO4 and with saturated aq NaHCO3, dried over Na2SO4, filtered,
and concentrated in vacuo. Purification by flash chromatography
(98:2, EtOAc/MeOH) afforded 20 (20 mg, 20% yield) as a foam: Rf
Notes
The authors declare no competing financial interest.
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