Application of the Ugi reaction with multiple amino acid-derived components: Synthesis and conformational evaluation of piperazine-based minimalist peptidomimetics

Article abstract of DOI:10.1039/c5ob00218d

The concurrent employment of α-amino acid-derived chiral components such as aldehydes and α-isocyanoacetates, in a sequential Ugi reaction/cyclization two-step strategy, opens the door to the synthesis of three structurally distinct piperazine-based scaff

Full text of DOI:10.1039/c5ob00218d

Organic &
Biomolecular Chemistry
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PAPER
Application of the Ugi reaction with multiple
amino acid-derived components: synthesis and
conformational evaluation of piperazine-based
minimalist peptidomimetics†
Cite this: Org. Biomol. Chem., 2015,
3, 4993
1
a
a
b
a
Mattia Stucchi, Silvia Cairati, Rengul Cetin-Atalay, Michael S. Christodoulou,
c
d
a
e
Giovanni Grazioso, Gennaro Pescitelli, Alessandra Silvani,* Deniz Cansen Yildirim
and Giordano Lesma
a
The concurrent employment of α-amino acid-derived chiral components such as aldehydes and α-iso-
cyanoacetates, in a sequential Ugi reaction/cyclization two-step strategy, opens the door to the synthesis
of three structurally distinct piperazine-based scaffolds, characterized by the presence of L-Ala and/or
L-Phe-derived side chains and bearing appropriate functionalities to be easily applied in peptide chemistry.
By means of computational studies, these scaffolds have been demonstrated to act as minimalist peptido-
mimetics, able to mimic a well defined range of peptide secondary structures and therefore potentially
useful for the synthesis of small-molecule PPI modulators. Preliminary biological evaluation of two
different resistant hepatocellular carcinoma cellular lines, for which differentiation versus resistance ability
seem to be strongly correlated with well defined types of PPIs, has revealed a promising antiproliferative
activity for selected compounds.
Received 2nd February 2015,
Accepted 19th March 2015
DOI: 10.1039/c5ob00218d
www.rsc.org/obc
A minor fraction of the protein–protein interface residues
can account for the majority of the free energy of binding
Introduction
Many proteins exert their biological roles as components of between proteins. Statistical analyses of structurally character-
complexes, and their functions are often determined by ized protein–protein interfaces have shown that side-chain
specific protein–protein interactions (PPIs). In the past decade, substituents account for about 80% of the interactions, with
2
PPIs have begun to gain attention as viable targets for thera- the polyamide backbone accounting for much less. Since it is
peutic intervention, despite the fact that they do not involve well known that the majority of PPIs are mediated by three
endogenous small molecule ligands that could provide leads main recognition motifs (α-helix, β-turn, or β-strand), an attrac-
for discovery programmes. In particular, the targeting of tive approach for the discovery of PPI modulators is to mimic
protein–protein interactions relevant to cancer is of fundamen- the key interaction residues using small molecule mimetics of
3
,4
tal importance, as the tumor-promoting function of several these recognition motifs.
aberrantly expressed proteins in the cancerous state is directly In recent years, an important development in peptide
correlated to their ability to interact with a protein-binding mimicry has been the emergence of analogues of peptide
1
partner.
secondary structures that mainly present selected side-chains,
with the main-chain polyamide backbone abbreviated or
totally absent. This approach is appealing because small mole-
cules without polyamide backbones are more likely to be orally
bioavailable and proteolytically stable. Compounds that
present only selected side-chains to resemble peptide second-
ary structures were referred to, for the first time, as minimalist
a
Dipartimento di Chimica, Università degli Studi di Milano, via Golgi 19, 20133
Milano, Italy. E-mail: alessandra.silvani@unimi.it; Tel: +39 0250314080
b
Cancer Systems Biology Laboratory, Graduate School of Informatics, METU,
0
c
6800 Ankara, Turkey
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano,
via L. Mangiagalli 25, 20133 Milano, Italy
5
mimics by Burgess and coworkers in 2011.
d
Dipartimento di Chimica e Chimica Industriale, Università di Pisa, via Moruzzi 3,
Minimalist peptidomimetics are likely to be most useful for
targets where exact binding conformations are unknown. In
these situations such compounds can have the intrinsic ability
to adjust conformations via rotation around a few significant
degrees of freedom, allowing such mimics to easily adapt.
5
e
6124 Pisa, Italy
Department of Molecular Biology and Genetics, Faculty of Science,
Bilkent University, Ankara, Turkey
Electronic supplementary information (ESI) available. See DOI: 10.1039/
†
c5ob00218d
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Minimalist mimics must be amenable to synthetic diversifica- preparation of isocyanides from α-amino acid ester hydro-
tion and conveniently accessible with side-chains that corres- chlorides, we selected a two-step sequence, involving formyl-
pond to the protein amino acids in order to be generally ation of the precursor by reaction with trimethyl orthoformate
useful. In this context, reactions that transform protein without the use of a solvent, followed by dehydration of the
amino acids into carbocyclic or heterocyclic backbones that obtained α-N-formylamino acid esters, by means of triphos-
have only a few significant degrees of freedom are particularly gene as a mild dehydrating agent and N-methyl morpholine as
2
0
useful.
a base. In this way, enantiomerically pure isocyanide com-
Following our interest in the multicomponent reaction ponents 3 and 4 have been prepared. The carboxylic acid and
(
MCR)/cyclization approach to the synthesis of conformation- the amine Ugi components were properly chosen as bifunc-
6
ally constrained peptidomimetics, in this work we demon- tional substrates in order to mediate highly selective outcomes
strate for the first time the application of this kind of strategy in the post-Ugi cyclization steps.
to the synthesis of minimalist peptidomimetics.
First of all, using chloroacetic acid and p-anisidine, we
7
The atom economy of MCRs, their convergent character, could access a small family of 2,5-diketopiperazine-based
operational simplicity, and the structural diversity and com- peptidomimetics, as reported in Scheme 1. The Ugi reaction was
plexity of the resulting molecules make this chemistry excep- conducted after a precondensation time of two hours between
tionally useful for drug discovery processes. In particular, Ugi- p-anisidine and the amino aldehyde (1 or 2), as suggested by
2
1
MCRs have undergone developments over the years, and Carney and coworkers in order to avoid the risk of loss of
various modifications in classic isocyanide-based multicompo- optical purity of the isocyanoacetates. Ugi compounds 5–8
nent reactions (IMCRs) have been achieved by the introduction were all obtained in good yields (87–91%), but as unseparable
8
of unusual building blocks, by transformation of the IMCR diastereoisomeric mixtures, with the exception of compounds
9
,10
products using post-condensation reactions
or by perform- 5, which could be separated by flash chromatography (d.r.
1
1–13
ing intramolecular IMCRs with bifunctional inputs.
5a : 5b 21 : 79). The two Ugi diastereoisomers 5a and 5b were
With regard to the synthesis of peptidomimetics, replace- separately cyclized by means of cesium carbonate in dry aceto-
1
4
2
2
ment of the amine or acid component in the Ugi reaction with nitrile, to afford 2,5-diketopiperazines 9a and 9b, whose
natural amino acids has had a number of successful out- overall stereochemistry has been assigned on the basis of com-
1
5,16
comes.
However, only a few reports make use of amino putational, NMR and CD studies (vide infra). Cyclization per-
acid-derived chiral isocyanides in IMCR, probably because of formed better for compound 5b, affording 9b in 76% yield,
the believed, but surmountable, configurationally instability of whereas it proved to be quite difficult for 5a, so that compound
1
7
chiral α-substituted isocyanoacetates. Moreover, the use of 9a could be isolated only in 16% yield, under heating. Evi-
amino acid-derived chiral aldehydes as the carbonyl com- dently, steric factors play a key role in the more or less smooth
1
8
ponent has almost not been documented at all, despite its achievement of a crowded trisubstituted 2,5-diketopiperazine
potential for enabling secondary reactions in order to con- ring. The same cyclization reaction was then applied to Ugi
strain or improve ‘drug-likeness’ of the initial flexible peptide- adducts 6–8 to afford the corresponding compounds 10–12,
like products.
which proved to be isolable as unique diastereoisomers in
By employing amino acid-derived chiral α-isocyanoacetates moderate yields (46–56%). This result can be rationalized in
and aldehydes, we pursued the synthesis of minimalist pepti- the light of the observed extremely different propensity of sep-
domimetics relying on three different piperazine-based cores, arated Ugi diastereoisomers 5 to cyclize to 9.
spanning side-chains corresponding to two chemically diverse
Starting again from aldehydes 1 and 2 and isocyanoacetates
amino acids, namely L-Ala and L-Phe. Piperazines and their 3 and 4 and varying the acid and amine components in the
keto analogs are considered privileged scaffolds in medicinal Ugi reaction, we could also access strictly related 2,6-diketo-
chemistry, thanks to their versatile binding properties and fre- piperazine-based peptidomimetics 17–20, as reported in
quent recurrence among positive hits encountered in biologi- Scheme 2.
cal screens.
In this case the Ugi reaction was performed employing
We also accomplished a computational evaluation of their acetic acid and glycine benzylester as the acid and amine com-
secondary structure mimicking properties and a biological ponent respectively, affording intermediates 13–16, with yields
1
screening within the COST Action CM 1106, entitled Chemical up to 89% and d.e. up to 84% (from H NMR). Catalytic
1
9
Approaches to Targeting Drug Resistance in Cancer Stem Cells.
removal of the benzylester group, followed by activation with
2
3
carbonyl diimidazole in tetrahydrofuran,
allowed us to
obtain the desired products 17–20 in good yields. Only for the
Ugi intermediate 13 it was possible to perform a chromato-
graphic separation of diastereoisomers (d.r. 13a : 13b
Results and discussion
As the carbonyl moiety for the Ugi reaction, we focused our 11.5 : 88.5) and achieve distinct compounds 17a and 17b, in
attention on enantiomerically pure N-methyl-N-Boc amino 15% and 59% yield respectively. Clearly, as for the above
aldehydes 1 and 2 (Scheme 1), easily prepared in good yields case of 2,5-diketopiperazines, also the cyclizations of Ugi
starting from the corresponding commercial N-Boc amino adducts 13a and 13b, to give the 2,6-diketopiperazines 17a
acids. Among the reported synthetic procedures for the and 17b, are subject to quite different steric restrictions.
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2 3 3
Scheme 1 Reagents and conditions: (a) p-anisidine, MeOH, rt, 2 h; then chloroacetic acid, 60 h. (b) Flash chrom. (c) Cs CO , 10% LiI, CH CN, rt (or
6
0 °C for 5a to 9a), 20 h.
2
Scheme 2 Reagents and conditions: (a) glycine benzylester, MeOH, rt, 2 h; then AcOH, 60 h. (b) Flash chrom. (c) H , 10% Pd/C, MeOH, rt, 2 h; then
CDI, THF, 75 °C to rt, 4 h.
2
,6-diketopiperazines 18–20 were obtained in good yields were employed in the Ugi reaction under usual conditions,
1
(
71–77%) and high d.e. (80–95%, from H NMR).
Finally, the two-step Ugi/cyclization strategy employing a amine and benzoic acid. The obtained Ugi products proved to
bifunctional amine was also applied to the synthesis of 3,4- be highly unstable and therefore were not isolated, but directly
dihydropyrazin-2(1H)-one-based peptidomimetics 23–26 cyclized to give final compounds 23–26 in good overall yields.
Scheme 3). In this case, N-Cbz amino aldehydes 21 and 22 In this case, all diastereoisomeric compounds 23a–26a and
together with isocyanoacetates 3 and 4, 2,2-diethoxyethan-
(
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Scheme 3 Reagents and conditions: (a) 2,2-diethoxyethanamine, MeOH, rt, 2 h; then benzoic acid, 60 h. (b) 50% TFA, DCM, rt, 24 h; then flash
chrom.
2
3b–26b could be easily separated by chromatography, allow-
ing us to estimate very high diastereoisomeric ratios, up to
b : a > 93 : 7.
In order to rationalize the stereochemical outcome of the
realized Ugi/cyclization processes, we relied on theoretical con-
formational analysis, NMR and CD spectra. Unfortunately, any
attempt to obtain crystals suitable for X-ray diffraction analysis
proved to be unsuccessful for all compounds in a wide range
of solvents and crystallization conditions.
We focused our analysis on compounds 9 and performed a
theoretical conformational search, which was run by the
Monte-Carlo algorithm and molecular mechanics (MMFF force
field), including all rotatable bonds and the puckering of
Fig. 1 Superposition of the first 8 low-energy structures of isomer
C-2(R) of compound 9, calculated at the B3LYP/6-31G(d) level.
6
-membered ring atoms. All structures thus obtained were geo-
metry-optimized with the DFT method at the B3LYP/6-31G(d)
level in vacuo. All structures obtained with population >1% at
room temperature were considered in the following. The calcu-
lations were run independently for the C-2(R) and C-2(S)
isomers of 9 (for atom numeration, see the structure in
Table 1).
The conformational analysis for the C-2(R) isomer revealed
a strong preference for conformations with anti orientation
between H-2 and H-15 and a pseudo-axial position of the C-2
appendage. Practically all populated structures at 300 K show a
consistent conformation around the C-2/C-15 bond (Fig. 1). In
this conformational family, aromatic ortho hydrogens are
expected to give NOE with the tBu group of the Boc moiety and
3 3
with NCH , but not with CH -17.
The conformational situation for the C-2(S) isomer is less
clear-cut. While the C-2 appendage occupies again a pseudo-
axial orientation, there are at least two main conformational
families, one (more favored, Fig. 2a) with gauche orientation
between H-2 and H-15 and a second (less favored, Fig. 2b) with
anti orientation. Although the NOE analysis is less straight-
forward, we can infer that aromatic ortho hydrogens are
expected to give a sizable NOE with CH -17, and smaller or no
NOE with the Boc moiety and NCH , respectively.
3
3
a
Table 1 Predicted and experimentally-observed diagnostic NOEs
Comparison of the expected NOEs based on molecular
modeling with the experimentally observed data obtained
from ROESY correlation peaks (Table 1) offers a strong indi-
cation to assign a C-2(S) configuration to the major diastereo-
isomer 9b and, conversely, a C-2(R) one to the minor
diastereoisomer 9a.
The configuration of the stereogenic carbon C-2 of the
major diastereoisomer 9b as C-2(S) was also studied by elec-
Predicted
for
C-2(S)
Predicted
for
C-2(R)
Exp. for 9b
(major
diast.)
Exp. for 9a
(minor
diast.)
2
4
tronic circular dichroism (CD). The CD spectrum of 9b,
measured in acetonitrile solution, displays a series of relatively
weak bands above 185 nm (ESI†). CD spectra were calculated
with the TDDFT method on DFT-optimized input structures
for the two C-2(R) and C-2(S) isomers. Unfortunately, the two
isomers led to a similar sequence of bands which in both
cases reproduces the experimental spectrum of 9b; therefore a
NOE
NCH
NCH
CH -17/H-27
3
3
/H-2
m
s
w
s
3
/H-27
—
m/s
w
m
—
s
—
m
—
m
—
m
tBu/H-27
a
Legend: s, strong; m, medium; w, weak; —, no NOE.
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Fig. 3 Proposed model for the diastereoselective formation of 5b in
Scheme 1.
and c were chosen as representative models of compounds 9b,
17b and 23b (Fig. 4), all characterized by the (S) stereochemical
Fig. 2 Superposition of the first 9 low-energy structures of isomer configuration on the piperazine-based ring and by L-Ala-
C-2(S) of compound 9, calculated at the B3LYP/6-31G(d) level (a, more
favored; b, less favored).
derived side chains. Models a, b and c contain the minimalist
core provided with N-Ac and CO-NHMe terminal groups,
aimed to mimic the insertion of the peptidomimetic into a
putative peptide.
safe discrimination based only on CD spectra is impossible
In this approach, the spatial mobility of the side chains
(ESI†). However, since CD spectroscopy does not contradict the connected to the central hetero-aromatic ring of the three
NMR results discussed above, we are confident of definitely models was analyzed by minimizing, equilibrating and heating
attributing the C-2(S) stereochemistry to the major diastereo- up the starting models to 300 K, 700 K and 1000 K for a short
isomer 9b and the C-2(R) one to the minor diastereoisomer 9a. period (2 ns). Finally, passing through an intermediate equili-
In accordance with this attribution, the observed diastereo- bration step at 700 K, 20 ns of MD simulations at room temp-
5
b
selectivity in the Ugi reaction (Scheme 1) can be rationalized erature (300 K) were performed.
The algorithm of the
2
5
by considering a Felkin–Ahn-like model, where the isocyano- AMBER12 package was executed for the entire calculation of
2
6
acetate reagent approaches towards the preformed iminium each model, using the GB implicit water solvent model.
intermediate from the less hindered side (Fig. 3), affording A frame every 10 ps of MD simulations was recorded, acquiring
preferentially 5b over 5a.
2800 conformational states of each mode (see the Experi-
The proposed model can also explain the trend of diastereo- mental section). Analyzing the dihedral angle fluctuation
selectivity which was observed for strictly related 6–8 Ugi (arrows in Fig. 4) over the obtained trajectories, the different
products. Reasoning that in all described Ugi/cyclization families of conformations for models a, b and c were
sequences (Schemes 2 and 3) the partial diastereocontrol is recognized.
likely to come from the addition of the isocyanoacetate reagent
Furthermore, the geometries representing those families of
2
7
to closely similar iminium intermediates, the reported conformations were energy minimized by GAUSSIAN09 at
diastereoselective outcomes could be reasonably explained by the quantum-mechanics DFT/B3LYP/6-31g(d)/CPCM-water
the application of Felkin–Ahn-like models similar to those level of calculation. The application of the Boltzmann equation
depicted in Fig. 3.
In order to assess our compounds as minimalist peptidomi- Fig. 4). Finally, in order to establish the correspondence of
provided the conformers distribution percentage (tables in
β
β
metics, a computational study was performed. Structures a, b C –C distances for peptidomimetics models a, b and c
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Fig. 4 Above: 2D structures of models a, b and c with their low energy conformers 3D images. Blue dotted line on model c represents the internal
β
β
hydrogen-bond. Below: tables reporting the Boltzmann distributions and the C –C distances.
with common secondary structures, the distances between
Table 2 Correspondence of C –Cβ distances for models a, b and c
β
the Cβ atoms of the side chains were measured (tables in with those reported for the most common peptide secondary structures.
Numbers highlighted by color represent the conformer percentage
showing the specific C –C distance/secondary structure. They were
retrieved from the Boltzmann distribution shown in Fig. 4
Fig. 4).
β
β
The results indicate that model b, in comparison with a
and c, possesses the highest number of thermodynamically
accessible conformers at room temperature. This can be due
to the minor steric hindrance of the N-substituent on the
piperazine core, with the acetyl group in model b allowing
major mobility for the vicinal side chains in comparison with
the 4-methoxy-phenyl and benzoyl groups of models a and c.
Comparing the C –C distances measured in the lowest
energy conformers of a, b and c with those reported by
Burgess and co-workers for typical secondary structures, we
β
β
5
were able to predict the secondary structures potentially
mimicked by our compounds (Table 2). Model compounds a
and c can assume conformations compatible with secondary
structures like α-helix, β-sheet (anti-parallel) and γ-turn
(classic). In contrast, the populated conformers of model b
may mimic all the secondary structures reported in Table 2.
Moreover, it has to be underlined that model c mimics pre-
dominantly the α-helix and the β-sheet (anti-parallel) second-
ary structures. This is due to an internal hydrogen-bond cells) and Mahlavu (PTEN deficient poorly differentiated cells),
stabilizing nearly only one structural geometry (Fig. 4).
for which differentiation versus resistance ability seems to be
2
8
Because of our particular interest in cancer drug resistance, strongly correlated with well defined types of PPIs. A signifi-
all compounds have been preliminarily evaluated for their cant antiproliferative effect, in the micromolar range (see
antiproliferative effects on two different hepatocellular carci- ESI†), has been observed for 12 and 25b and deserves further
noma (HCC) cellular lines, namely Huh7 (well differentiated studies, which are currently in progress.
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propanoate, 5b. Prepared according to the above general pro-
cedure from aldehyde 1 and isocyanide 3; FC: ethyl acetate–
Conclusion
We have demonstrated the feasibility of a two-step multi- n-hexane, 1 : 1.5; yield: 5a (97 mg, 19%), 5b (356 mg, 71%). 5a:
3
0
component synthesis of three complex, yet structurally distinct, colorless oil; R
f
0.21 (1.5 : 1 n-hexane–EtOAc); [α]
D
−16.3 (c 0.5,
1
piperazine-based scaffolds, in which bifunctional substrates CHCl ); H NMR (300 MHz, CD CN, 1 : 1 mixture of rotamers)
3
3
mediate highly selective outcomes based on Ugi/cyclization δ 7.38–7.08 (m, 3H), 6.95 (d, J = 8.8 Hz, 2H), 4.45–4.09 (br, s,
sequences. All scaffolds are characterized by the presence of 1H), 3.85 (br, s, 2H), 3.81 (s, 3H), 3.82–3.75 (m, 1H), 3.69 (s,
L-Ala and/or L-Phe-derived amino acid side chains and have 1.5H), 3.71–3.63 (m, 1H), 3.67 (br, s, 1.5H), 2.65 (s, 3H),
1
3
been demonstrated to act as minimalist peptidomimetics, able 1.46–1.31 (m, 12H), 1.27 (br, d, J = 6.8 Hz, 3H); C NMR
to mimic a range of secondary structures and therefore poten- (75 MHz, CD CN) δ 178.1, 173.0 (2C), 165.4, 160.2, 136.5,
3
tially useful for the synthesis of small-molecule PPI 136.2, 135.5, 120.0, 119.8, 84.8 and 84.6 (1C), 60.6, 60.5, 57.2,
modulators. Preliminary biological evaluation of cancer resist- 53.5, 53.4, 48.5, 48.3, 34.3, 33.0 (3C), 22.2 and 21.9 (1C), 19.7
+
+
ant cellular lines has revealed a promising antiproliferative and 19.6 (1C); HRMS (ESI) calcd for C23
3 7
H34ClN NaO [MNa]
activity for selected compounds, for which further investi- 522.1977, found 522.1983. 5b: colorless oil; R 0.18 (1.5 : 1
f
3
0
1
gation is currently underway.
n-hexane–EtOAc); [α]
CD CN, 1 : 1 mixture of rotamers) δ 7.40–7.08 (br, m, 1H), 7.20
m, 2H), 6.95 (d, J = 9.1 Hz, 2H), 4.41–4.12 (br, m, 1H), 3.86
D 3
+8.5 (c 0.5, CHCl ); H NMR (300 MHz,
3
(
(
(
1
br, s, 2H), 3.81 (s, 3H), 3.79 (m, 1H), 3.67 (br, s, 1.5H), 3.65
m, 1H), 3.64 (s, 1.5H), 2.67 (s, 3H), 1.38 (s, 9H), 1.32 (m, 3H),
Experimental section
General information
1
3
.27 (br, d, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl ) δ 173.5
3
All commercial materials (Aldrich, Fluka) were used without
further purification. All solvents were of reagent grade or
HPLC grade. All reactions were carried out under a nitrogen
atmosphere unless otherwise noted. All reactions were moni-
tored by thin layer chromatography (TLC) on precoated silica
gel 60 F254; spots were visualized with UV light or by treat-
and 173.4 (1C), 169.4–168.7 (2C), 160.6, 157.0 and 156.8 (1C),
131.4, 130.8, 130.2, 115.7, 115.5, 81.0 and 80.6 (1C), 56.1 (2C),
5
3.0, 52.9, 48.9 (2C), 43.1 and 43.0 (1C), 29.1 (4C), 18.2, 16.1;
+
+
HRMS (ESI) calcd for C23
found 522.1961.
H
34ClN
3
NaO
7
[MNa] 522.1977,
(
2S)-Methyl 2-((3S)-3-((tert-butoxycarbonyl)(methyl)amino)-
ment with a 1% aqueous KMnO
4
solution. Products were puri-
2
-(2-chloro-N-(4-methoxyphenyl)acetamido)butanamido)-3-
fied by flash chromatography on silica gel 60 (230–400 mesh).
phenylpropanoate, 6. Prepared according to the above general
procedure from aldehyde 1 and isocyanide 4; FC: ethyl acetate–
n-hexane, 1 : 1.5; yield 524 mg, (91%) as an inseparable
1
13
H NMR spectra and C NMR spectra were recorded on 300
and 400 MHz spectrometers. Chemical shifts are reported in
13
parts per million relative to the residual solvent. C NMR
spectra have been recorded using the APT pulse sequence.
mixture of diastereoisomers (d.e. 50%, NMR analysis): pale
1
yellow oil; R 0.24 (1.5 : 1 n-hexane–EtOAc); H NMR (300 MHz,
f
1
Multiplicities in H NMR are reported as follows: s = singlet,
3
CD CN, rotameric mixture of a mixture of diastereoisomers)
d = doublet, t = triplet, m = multiplet, br s = broad singlet. High-
resolution MS spectra were recorded with an FT-ICR (Fourier
Transform Ion Cyclotron Resonance) instrument, equipped
with an ESI source. UV-Vis spectra were recorded with a Jasco
V-650 spectrophotometer. CD spectra were recorded with a
JASCO J-715 spectropolarimeter; the conditions are reported in
the legend of Fig. 2.
δ 7.47–6.99 (m, 8H), 6.89 (br, d, J = 8.8 Hz, 2H), 4.80–4.40 (br,
m, 1H), 3.81 (br, s, 2H), 3.80–3.74 (m, 4H), 3.69 (s, 0.5H),
3.69–3.63 (m, 3.5H), 3.24–3.05 (br, m, 1H), 3.03–2.80 (br, m,
1
3
H), 2.62 (s, 2.75H), 2.58 (s, 0.25H), 1.36 (s, 9H), 1.28–1.21 (m,
+
+
H); HRMS (ESI) calcd for C H ClN NaO [MNa] 598.2290,
29 38 3 7
found 598.2305.
(
2S)-Methyl 2-((3S)-3-((tert-butoxycarbonyl)(methyl)amino)-
-(2-chloro-N-(4-methoxyphenyl)acetamido)-4-phenylbutan-
amido)propanoate, 7. Prepared according to the above general
2
General procedure for the synthesis of compounds 5–8
Aldehyde (1 or 2) (1.0 mmol, 1 eq.) was dissolved in 1 mL of procedure from aldehyde 2 and isocyanide 3; FC: ethyl acetate–
dry methanol under nitrogen, 4-methoxy aniline (1.0 mmol, n-hexane, 1 : 1.5; yield 501 mg, (87%) as an inseparable
1
23 mg, 1 eq.) was added and the resulting mixture was kept mixture of diastereoisomers (d.e. 56%, NMR analysis): pale
1
f
under stirring for 2 h at room temperature. 2-Chloroacetic acid yellow oil; R 0.22 (1.5 : 1 n-hexane–EtOAc); H NMR (300 MHz,
(
1.0 mmol, 94.5 mg, 1 eq.) and isocyanide (3 or 4) (1.2 mmol, CD CN, rotameric mixture of a mixture of diastereoisomers)
3
1
.2 eq.) were sequentially added and the reaction was stirred δ 7.40–7.04 (m, 8.4H), 6.96 (br, d, J = 8.4 Hz, 1.6H), 4.59–4.40
for an additional 60 h at room temperature. The resulting (br, m, 1H), 3.93–3.64 (m, 10H), 3.33–2.87 (m, 1H), 2.80 (s,
mixture was then concentrated under reduced pressure, to give 0.66H), 2.76 (s, 2.34H), 2.64 (m, 1H), 1.52–1.26 (m, 12H);
+
+
a residue which was purified by flash chromatography (FC) as HRMS (ESI) calcd for C29
indicated below. found 598.2305.
S)-Methyl-2-((2R,3S)-3-((tert-butoxycarbonyl)(methyl)amino)- (2S)-Methyl 2-((3S)-3-((tert-butoxycarbonyl)(methyl)amino)-
-(2-chloro-N-(4-methoxyphenyl)acetamido)butanamido)propanoate, 2-(2-chloro-N-(4-methoxyphenyl)acetamido)-4-phenylbutan-
a and (S)-methyl-2-((2S,3S)-3-((tert-butoxycarbonyl)(methyl)- amido)-3-phenylpropanoate, 8. Prepared according to the
amino)-2-(2-chloro-N-(4-methoxyphenyl)acetamido)butanamido)- above general procedure from aldehyde 2 and isocyanide 4;
H
38ClN
3
NaO
7
[MNa] 598.2290,
(
2
5
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FC: ethyl acetate–n-hexane, 1 : 1.5; yield 573 mg, (88%) as an 5.68 (dd, J = 11.4, 5.1 Hz, 1H), 4.70 (br, m, 1H), 4.41 (d, J =
inseparable mixture of diastereoisomers (d.e. 51%, NMR analy- 17.3 Hz, 1H), 4.03 (d, J = 17.3 Hz, 1H), 3.88 (br, m, 1H), 3.81 (s,
1
sis): oil; R
CDCl
7
1
2
f
0.44 (1.5 : 1 n-hexane–EtOAc); H NMR (400 MHz, 3H), 3.80 (s, 3H), 3.40 (dd, J = 14.4 and 5.1 Hz, 1H), 2.99 (dd,
3
, rotameric mixture of a mixture of diastereoisomers) δ J = 14.4, 11.4 Hz, 1H), 2.68 (s, 3H), 1.46 (s, 9H), 1.17 (d, J =
.38–7.06 (m, 11.4H), 7.02–6.80 (br, m, 3.6H), 5.05–4.67 (br, m, 7.1 Hz, 3H); C NMR (100 MHz, CDCl ) δ 170.7, 166.8, 164.7,
H), 3.93–3.58 (m, 10H), 3.32–3.18 (m, 1H), 3.05–2.86 (m, 2H), 159.3, 156.7, 136.1, 133.0, 129.8 (2C), 129.4 (2C), 129.2 (2C),
1
3
3
.78 (s, 0.75H), 2.75 (s, 2.25H), 2.65 (m, 1H), 1.40 (m, 9H); 127.7, 115.0 (2C), 80.9, 69.0, 56.6, 56.1, 53.0, 51.9, 47.6, 35.5,
+
+
+
HRMS (ESI) calcd for C H ClN NaO [MNa] 674.2603, 29.9, 29.1 (3C), 17.1; HRMS (ESI) calcd for C H N NaO
7
3
5
42
3
7
29 37
3
+
found 674.2622.
[MNa] 562.2524, found 562.2509.
S)-Methyl-2-((S)3-((S)-1-((tert-butoxycarbonyl)(methyl)amino)-
-phenylethyl)-4-(4-methoxyphenyl)-2,5-dioxopiperazin-1-yl)-
To a solution of the Ugi product (5a, 5b, 6, 7 or 8) (0.5 mmol, propanoate, 11. Prepared according to the above general pro-
eq.) in dry acetonitrile (4.5 mL) under nitrogen, cesium car- cedure from 7; FC: ethyl acetate–n-hexane, 1 : 1.5; yield:
bonate (1 mmol, 326 mg, 4 eq.) and LiI (0.05 mmol, 7 mg, 0.1 178 mg (66%); pale yellow oil; R 0.15 (1.5 : 1 n-hexane–EtOAc);
(
General procedure for the synthesis of compounds 9–12
2
1
f
3
0
1
eq.) were added, and the mixture was stirred for 20 h at room [α]
D 3 3
+2.2 (c 0.5, CHCl ); H NMR (400 MHz, CDCl ) δ 7.35–7.24
temperature (or at 60 °C, for 5a). The mixture was quenched (m, 2H), 7.24–7.12 (m, 3H), 7.12–7.05 (m, 2H), 6.92 (d, J =
with satd aq. NH Cl (20 mL) and extracted with EtOAc (3 × 8.6 Hz, 2H), 5.29 (q, J = 7.1 Hz, 1H), 4.97 (br, m, 1H), 4.43–4.30
4
2
0 mL). The combined organic layers were washed with brine, (m, 2H), 3.99 (d, J = 16.7 Hz, 1H), 3.83 (s, 3H), 3.74 (s, 3H),
dried over Na SO and concentrated under reduced pressure, 3.08 (br, m, 1H), 2.77 (br, m, 2H), 2.67 (s, 3H), 1.47 (d, J =
to give a residue, which was purified by flash chromatography 7.1 Hz, 3H), 1.44 (s, 9H); C NMR (100 MHz, CDCl ) δ 171.8,
2
4
1
3
3
(FC) as indicated below.
166.0, 164.7, 159.3, 157.1, 137.7, 132.9, 129.6 (2C), 129.1 (2C),
(S)-Methyl 2-((R)-3-((S)-1-((tert-butoxycarbonyl)(methyl)- 128.8 (2C), 127.2, 115.1 (2C), 81.0, 67.3, 57.7, 56.1, 52.3, 51.7,
amino)ethyl)-4-(4-methoxyphenyl)-2,5-dioxopiperazin-1-yl)pro-
panoate, 9a. Prepared according to the above general pro-
cedure from 5a; FC: ethyl acetate–n-hexane, 1 : 1.5; yield: 37 mg
47.6, 36.9, 30.8, 28.9 (3C), 14.5; HRMS (ESI) calcd for
+
+
C
29
H
37
N
3
7
NaO [MNa] 562.2524, found 562.2536.
(2S)-Methyl-2-((S)3-((S)-1-((tert-butoxycarbonyl)(methyl)amino)-
(
16%); colorless oil; Rf 0.18 (1.5 : 1 n-hexane–EtOAc); 2-phenylethyl)-4-(4-methoxyphenyl)-2,5-dioxopiperazin-1-yl)-
3
0
1
[α]
D
3 3
−12.7 (c 0.5, CHCl ); H NMR (300 MHz, CD CN) δ 7.43 3-phenylpropanoate, 12. Prepared according to the above
(br, m, 2H), 6.98 (d, J = 8.8 Hz, 2H), 5.03 (q, J = 7.0 Hz, 1H), general procedure from 8; FC: ethyl acetate–n-hexane, 3 : 7;
4
3
1
.63 (br, m, 1H), 4.34 (br, m, 1H), 4.30 (br, d, J = 16.6 Hz, 1H), yield: 151 mg (56%); yellow oil; R 0.21 (7 : 3 n-hexane–EtOAc);
f
3
1
1
.88 (d, J = 16.7 Hz, 1H), 3.83 (s, 3H), 3.73 (s, 3H), 2.67 (s, 3H), [α]
D
3
= −74.7 (c 1.0, MeOH); H NMR (400 MHz, CDCl ) δ
.45 (s, 9H), 1.44 (d, J = 7.0 Hz, 3H), 1.24 (br, d, J = 7.5 Hz, 3H); 7.35–7.23 (m, 4H), 7.23–7.18 (m, 2H), 7.18–7.11 (m, 2H),
1
3
C NMR (100 MHz, CD CN) δ 172.2, 168.4, 165.0 (2C), 160.7, 7.10–6.99 (m, 4H), 6.88 (d, J = 8.8 Hz, 2H), 5.73 (dd, J = 11.3
3
1
4
33.9, 131.4 (2C), 130.0, 115.3 (2C), 80.9, 68.5, 55.8, 52.5 (2C), and 5.3 Hz, 1H), 4.94 (br, m, 1H), 4.40 (d, J = 17.2 Hz, 1H),
9.1 and 48.9 (1C), 48.0, 28.3 (3C), 17.4, 14.2; HRMS (ESI) 4.05 (d, J = 17.2 Hz, 1H), 4.03 (br, m, 1H), 3.83 (s, 3H), 3.81 (s,
+
+
calcd for C H N NaO [MNa] 486.2211, found 486.2227.
3H), 3.44 (dd, J = 14.4 and 5.3 Hz, 1H), 3.03 (dd, J = 14.4 and
2
3
33
3
7
1
3
(
S)-Methyl
2-((S)-3-((S)-1-((tert-butoxycarbonyl)(methyl)- 11.3 Hz, 1H), 3.05–2.77 (m, 2H), 2.64 (s, 3H), 1.40 (s, 9H);
C
amino)ethyl)-4-(4-methoxyphenyl)-2,5-dioxopiperazin-1-yl)pro-
NMR (100 MHz, CDCl ) δ 170.6, 166.8, 164.7, 159.5, 157.0,
3
panoate, 9b. Prepared according to the above general pro- 137.5, 136.1, 132.2, 129.7 (2C), 129.4–129.0 (8C), 127.8, 127.1
cedure from 5b; FC: ethyl acetate–n-hexane, 1 : 1.5; yield: (2C), 115.1, 80.9, 67.1, 56.7, 56.1 (2C), 53.1, 47.4, 36.7, 35.4,
+
+
1
76 mg (76%); colorless oil; R
f
0.16 (1.5 : 1 n-hexane–EtOAc); 30.3, 28.9 (3C); HRMS (ESI) calcd for C35
41 3 7
H N NaO [MNa]
3
0
1
[
(
α] +24.2 (c 1.0, MeOH); H NMR (400 MHz, CD CN) δ 7.36 638.2837, found 638.2847.
br, d, J = 8.9 Hz, 2H), 6.98 (d, J = 9.2 Hz, 2H), 5.12 (q, J = 7.3
D
3
Hz, 1H), 4.73 (br, m, 1H), 4.29 (d, J = 16.7 Hz, 1H), 4.27 (br, m,
H), 3.88 (br, d, J = 16.7 Hz, 1H), 3.83 (s, 3H), 3.69 (s, 3H), 2.68
s, 3H), 1.46 (d, J = 7.3 Hz, 3H), 1.45 (s, 9H), 1.16 (br, d, J = 7.0 Aldehyde (1 or 2) (1.0 mmol, 1 eq.) was dissolved in 1 mL of
General procedure for the synthesis of compounds 13–16
1
(
1
3
Hz, 3H); C NMR (100 MHz, CD
1
5
3
CN) δ 172.2, 165.9, 164.9, dry methanol under nitrogen, O-benzyl glycine (1.0 mmol,
59.1, 133.9, 130.0, 129.2 (2C), 114.6 (2C), 80.2, 68.4, 55.9, 165 mg, 1 eq.) was added and the resulting mixture was kept
2.7, 52.1, 51.5, 48.0, 29.5, 28.3 (3C), 16.5, 13.8; HRMS (ESI) under stirring for 2 h at room temperature. Acetic acid
+
+
calcd for C23
H
33
N
3
NaO
7
[MNa] 486.2211, found 486.2216.
(1.0 mmol, 60 mg, 1 eq.) and isocyanide (3 or 4) (1.2 mmol, 1.2
(S)-Methyl-2-((S)3-((S)-1-((tert-butoxycarbonyl)(methyl)amino)- eq.) were sequentially added and the reaction was stirred for
ethyl)-4-(4-ethoxyphenyl)-2,5-dioxopiperazin-1-yl)-3-phenyl- an additional 60 h at room temperature. The resulting mixture
propanoate, 10. Prepared according to the above general pro- was diluted with water (10 mL) and extracted with EtOAc (3 ×
cedure from 6; FC: ethyl acetate–n-hexane, 3 : 7; yield: 124 mg 5 mL). The combined organic layers were dried over Na SO
2
4
2
2
(
46%); wax; R
f
0.15 (7 : 3 n-hexane–EtOAc); [α]
D
−55.2 (c 1.0, and the solvent was removed under reduced pressure to afford
1
CHCl
3
); H NMR (400 MHz, CDCl
3
) δ 7.34–7.23 (m, 3H), the crude Ugi product, which was purified by flash chromato-
7
.21–7.16 (m, 2H), 7.14–7.05 (m, 2H), 6.90 (d, J = 8.9 Hz, 2H), graphy (FC) as indicated below.
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(
S)-Methyl
2-((2R,3S)-2-(N-(2-(benzyloxy)-2-oxoethyl)acet- 2.00 (br, s, 2.76H), 1.97 (s, 0.24H), 1.43 (br, s, 9H), 1.36 (br, m,
+
+
amido)-3-((tert-butoxycarbonyl)(methyl)amino)butanamido)- 3H); HRMS (ESI) calcd for C H N NaO [MNa] 606.2786,
3
1
41
3
8
propanoate, 13a and (S)-methyl 2-((2S,3S)-2-(N-(2-(benzyloxy)-2- found 606.2761.
oxoethyl)acetamido)-3-((tert-butoxycarbonyl)(methyl)amino)- (2S)-Methyl-2-((3S)-2-(N-(2-(benzyloxy)-2-oxoethyl)acetamido)-
butanamido)propanoate, 13b. Prepared according to the 3-((tert-butoxycarbonyl)(methyl)amino)-4-phenylbutanamido)-
above general procedure from aldehyde 1 and isocyanide 3; 3-phenylpropanoate, 16. Prepared according to the above
FC: ethyl acetate–n-hexane, 1 : 1.5; yield: 13a (35 mg, 7%), 13b general procedure from aldehyde 2 and isocyanide 4; FC: ethyl
(
276 mg, 54%). 13a: amber oil; R 0.18 (1 : 1.5 n-hexane–EtOAc); acetate–n-hexane, 1 : 1.5; yield: 587 mg (89%), as an insepar-
f
3
0
1
[α]
D
−3.4 (c 0.5, CHCl
: 1 mixture of rotamers) δ 7.34 (br, m, 5H), 6.62–6.27 (br, m, colorless oil;
3
); H NMR (300 MHz, CDCl
3
,
able mixture of diastereoisomers (d.e. 72%, NMR analysis):
1
1
1
4
R
f
0.18 (1.5 : 1 n-hexane–EtOAc);
H NMR
H), 5.16–5.08 (m, 2H), 5.06 (br, m, 1H), 4.79–4.61 (br, m, 1H), (300 MHz, CDCl , rotameric mixture of two diastereoisomers)
3
.47 (d, J = 19.5 Hz, 0.5H), 4.45 (d, J = 19.5 Hz, 0.5H), 4.23 (br, δ 7.43–7.01 (m, 16H), 5.29–5.09 (m, 3H), 4.88 (m, 0.86H), 4.74
m, 1H), 4.01 (br, d, J = 19.5 Hz, 1H), 3.72 (s, 1.5), 3.71 (s, 1.5), (m, 0.14H), 4.64–4.43 (m, 1.86H), 4.29 (m, 0.14H), 4.01 (br, d,
.62 (br, s, 3H), 1.98 (br, s, 1.5), 1.95 (s, 1.5), 1.42 (s, 9H), 1.36 J = 16.6 Hz, 1H), 3.74 (s, 0.24H), 3.67 (s, 1.26H), 3.65 (s, 1.5H),
2
1
3
(d, J = 6.8 Hz, 3H), 1.14 (br, m, 3H); C NMR (75 MHz, CDCl
3
,
3.54–2.84 (m, 4H), 2.70 (s, 2.52H), 2.63 (s, 0.48H), 2.07 (s,
mixture of rotamers) δ 173.0 and 172.9 (1C), 172.6 and 172.4 0.24H), 1.98 (s, 1.5H), 1.95 (s, 1.26H), 1.41 (s, 9H); HRMS (ESI)
+
+
(
1C), 168.2 and 168.1 (1C), 167.8, 155.8, 135.4, 128.6–128.2 calcd for C H N NaO [MNa] 682.3099, found 682.3114.
37 45 3 8
(
4
5C), 79.4, 66.9, 57.4–56.0 (br, 1C), 52.3, 48.4, 48.1, 47.1 and
6.7 (br, 1C), 28.7, 28.4 (3C), 21.6, 18.0, 15.2 and 14.8 (1C);
General procedure for the synthesis of compounds 17–20
+
+
HRMS (ESI) calcd for C H N NaO [MNa] 530.2473, found Palladium (10 wt% on carbon, 70 mg) was added to a solution
2
5
37
3
8
3
0
5
30.2455. 13b: R
f
0.15 (1 : 1.5 n-hexane–EtOAc); [α]
); H NMR (300 MHz, CDCl , 1 : 1 mixture of confor- in methanol (3 mL). The reaction mixture was degassed
mers) δ 7.35 (br, m, 5H), 6.65–6.28 (br, m, 1H), 5.17 (br, m, in vacuo, placed under an atmosphere of H (g), and stirred in
D
+8.8 (c 0.5, of the Ugi product (13a, 13b, 14, 15 or 16) (0.30 mmol, 1 eq.)
1
CHCl
3
3
2
3
3
H), 4.51–4.19 (br, m, 4H), 3.70 (s, 1.5), 3.68 (s, 1.5), 2.81 (s, the dark at rt for 2 h. The mixture was filtered through a pad
H), 2.03 (br, s, 3H), 1.42 (s, 9H), 1.32 (d, J = 6.8 Hz, 3H), 1.15 of Celite eluting with methanol (10 mL), and the combined
1
3
(
(
1
1
5
d, J = 6.8 Hz, 1.5H), 1.12 (d, J = 6.8 Hz, 1.5H); C NMR organic layers were concentrated in vacuo to give the crude
75 MHz, CDCl , mixture of rotamers) δ 172.4–172.0 (2C), carboxylic acid intermediate, which was directly used in the
69.6, 168.9 and 168.8 (1C), 155.4 and 155.2 (1C), 135.4 and next step, as follows. 1,1′-Carbonyl diimidazole (0.30 mmol,
35.2 (1C), 129.1–128.0 (5C), 80.3, 67.4 and 67.2 (1C), 59.6, 127 mg, 1 eq.) was added to a solution of the crude carboxylic
2.6, 48.5, 48.1, 47.8, 29.7, 28.4 (3C), 21.6, 17.6, 15.7 and 15.3 acid in dry tetrahydrofuran (3 mL), under nitrogen, and the
3
+
+
(1C); HRMS (ESI) calcd for C25
H
37
N
3
NaO
8
[MNa] 530.2473, resulting mixture was refluxed for 60 min and then stirred for
found 530.2482.
an additional 3 h at room temperature. The solvent was
(
2S)-Methyl-2-((3S)-2-(N-(2-(benzyloxy)-2-oxoethyl)acetamido)- removed under reduced pressure to give a residue which was
-((tert-butoxycarbonyl)(methyl)amino)butanamido)-3-phenyl- partitioned between CHCl (10 mL) and 1 M HCl (10 mL). The
propanoate, 14. Prepared according to the above general pro- aqueous phase was extracted with CHCl (3 × 10 mL) and the
3
3
3
cedure from aldehyde 1 and isocyanide 4; FC: ethyl acetate–n- combined organic layers were washed with H
hexane, 1 : 1; yield: 344 mg (59%), as an inseparable mixture of Na SO and concentrated in vacuo to afford the crude product,
diastereoisomers (d.e. 80%, NMR analysis): pale yellow oil; Rf which was purified by flash chromatography (FC), as indicated
2
O, dried over
2
4
1
0
.27 (1 : 1 n-hexane–EtOAc); H NMR (300 MHz, CDCl
meric mixture of two diastereoisomers) δ 7.41–7.03 (m, 11H),
.28–5.10 (m, 3H), 4.78 (br, m, 0.9H), 4.78–3.93 (m, 3.1H), 3.68 (methyl)amino)ethyl)-2,6-dioxopiperazin-1-yl)propanoate, 17a.
3
, rota- below.
(S)-Methyl
2-((R)-4-acetyl-3-((S)-1-((tert-butoxycarbonyl)-
5
(s, 0.3H), 3.65 (s, 2.7H), 3.09 (br, m, 1H), 3.01 (m, 1H), 2.75 Prepared according to the above general procedure from 13a;
(
1
C
br, s, 2.7H), 2.58 (s, 0.3H), 1.97 (br, m, 3H), 1.42 (s, 0.9H), FC: ethyl acetate–n-hexane, 7 : 3; yield: 18 mg (15%); colorless
3
0
1
.39 (s, 8.1H), 1.09 (br, d, J = 6.8 Hz, 3H); HRMS (ESI) calcd for oil; R 0.33 (3 : 7 n-hexane–EtOAc); [α] −15.6 (c 0.5, CHCl ); H
f
D
3
+
+
31
H
41
N
3
NaO
8
[MNa] 606.2786, found 606.2800.
3
NMR (300 MHz, CDCl ) δ 4.63–3.90 (m, 5H), 3.72 (br, s, 3H),
(
2S)-Methyl-2-((3S)-2-(N-(2-(benzyloxy)-2-oxoethyl)acetamido)- 2.82 (br, s, 3H), 2.08 (br, s, 3H), 1.45 (s, 9H), 1.37 (br, d, J = 6.8
1
3
3
-((tert-butoxycarbonyl)(methyl)amino)-4-phenylbutanamido)- Hz, 3H), 1.12 (br, d, J = 6.8 Hz, 3H); C NMR (100 MHz,
propanoate, 15. Prepared according to the above general CDCl ) δ 170.1, 169.1, 167.0, 166.5 and 166.4 (1C), 155.9, 80.5,
3
procedure from aldehyde 2 and isocyanide 3; FC: ethyl acetate– 55.0, 52.5, 49.5, 48.8, 46.5, 29.0, 28.3 (3C), 21.1, 15.5 and 15.4
+
n-hexane, 1.5 : 1; yield: 256 mg (44%), as an inseparable (1C), 14.2 and 13.9 (1C); HRMS (ESI) calcd for C H N NaO
1
8
29
3
7
+
mixture of diastereoisomers (d.e. 84%, NMR analysis): pale [MNa] 422.1898, found 422.1924.
1
yellow oil; R
f
0.20 (1 : 5 n-hexane–EtOAc); H NMR (300 MHz,
(S)-Methyl
(methyl)amino)ethyl)-2,6-dioxopiperazin-1-yl)propanoate, 17b.
.46–7.04 (m, 11H), 5.34–5.10 (m, 3H), 4.88 (br, m, 0.9H), Prepared according to the above general procedure from 13b;
.62–3.91 (m, 3.1H), 3.73 (s, 0.24H), 3.65 (s, 2.76H), 3.32 (br, FC: ethyl acetate–n-hexane, 7 : 3; yield: 71 mg (59%); colorless
2-((S)-4-acetyl-3-((S)-1-((tert-butoxycarbonyl)-
CDCl3, rotameric mixture of two diastereoisomers)
δ
7
4
3
0
1
m, 1H), 3.07 (br, m, 1H), 2.81 (br, s, 2.76H), 2.65 (s, 0.24H), oil; R 0.29 (3 : 7 n-hexane–EtOAc); [α] +39.9 (c 0.5, CHCl ); H
f
D
3
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1
3
3
NMR (400 MHz, CDCl , mixture of conformers 1.5 : 1) δ 5.36 (s, 2.7H), 2.08 (s, 0.3H), 1.45 (br, s, 9H); C NMR (101 MHz,
(
br, d, J = 9.9 Hz, 1H), 5.19 (m, 1H), 4.72 (m, 1H), 4.57 (d, J = CD CN), δ 169.7–168.5 (3C), 167.8 and 167.6 (1C), 154.7 and
3
1
0
2
7
8.7 Hz, 0.6H), 4.52 (d, J = 18.7 Hz, 0.4H), 4.29 (d, J = 18.7 Hz, 154.6 (1C), 138.3–137.8 (1C), 137.2–137.0 (1C), 129.1 (4C),
.4H), 4.24 (d, J = 18.7 Hz, 0.6H), 3.68 (s, 1.8H), 3.67 (s, 1.2H), 128.4 and 128.3 (4C), 126.7–126.4 (2C), 79.7–79.4 (1C), 56.2,
.82 (br, s, 1.8H), 2.80 (s, 1.2H), 2.23–2.15 (m, 3H), 1.52 (d, J = 54.9–54.6 (1C), 53.8, 52.1 and 52.0 (1C), 46.8 and 46.5 (1C),
.0 Hz, 1.8H), 1.45 (d, J = 7.0 Hz, 1.2H), 1.44–1.38 (m, 9H), 1.19 34.4–34.2 (2C), 27.9, 27.7 (3C), 21.0 and 20.8 (1C); HRMS (ESI)
1
3
+
+
3 37 3 7
(br, d, J = 6.5 Hz, 3H); C NMR (100 MHz, CDCl ) δ 169.9, calcd for C30H N NaO [MNa] 574.2524, found 574.2506.
1
2
C
69.0, 167.0, 166.4, 156.1, 80.2, 54.7, 52.4, 49.4, 48.7, 46.8,
General procedure for the synthesis of compounds 23–26
8.9, 28.3 (3C), 21.4, 15.5, 14.1; HRMS (ESI) calcd for
+
+
18
H
29
N
3
NaO
7
[MNa] 422.1898, found 422.1917.
Aldehyde (21 or 22) (1.0 mmol, 1 eq.) was dissolved in 1 mL of
(2S)-Methyl-2-(4-acetyl-3-((S)-1-((tert-butoxycarbonyl)(methyl)- dry methanol under nitrogen, 2,2-diethoxyethanamine
amino)ethyl)-2,6-dioxopiperazin-1-yl)-3-phenylpropanoate, (1.0 mmol, 133 mg, 1 eq.) was added and the resulting mixture
1
1
8. Prepared according to the above general procedure from was kept under stirring for 2 h at room temperature. Benzoic
4; FC: ethyl acetate–n-hexane, 1.5 : 1; yield: 105 mg (74%), as acid (1.0 mmol, 122 mg, 1 eq.) and isocyanide (3 or 4)
an inseparable mixture of diastereoisomers (d.e. 80%, NMR (1.2 mmol, 1.2 eq.) were sequentially added and the reaction
1
analysis); foam; R
f
0.36 (1 : 1.5 n-hexane–EtOAc); H NMR was stirred for an additional 24 h at room temperature. The
mixture of two diastereoisomers) solvent was removed under reduced pressure to afford the
δ 7.34–7.03 (m, 5H), 5.56–5.37 (br, m, 1.9H), 4.59 (br, m, 1H), unstable crude Ugi product, which was directly used in the
(
400 MHz, CD CN,
a
3
4
3
2
.57 (d, J = 18.7 Hz, 1H), 4.83–4.11 (m, 2H), 3.66 (br, s, 3H), next step. The crude was dissolved in 4.5 mL of 50% trifluoro-
.43 (m, 1H), 3.17–2.99 (m, 1H), 2.79 (s, 0.3H), 2.77 (s, 2.7H), acetic acid in dichloromethane, and the resulting solution was
1
3
.23 (s, 2.7H), 2.14 (s, 0.3H), 1.42 (s, 9H), 1.14 (m, 3H);
C
kept under stirring for 24 h. The solvent was removed under
NMR (101 MHz, CD
3
CN), δ 170.2–167.8 (4C), 156.3 and 156.0 reduced pressure to give a residue which was dissolved in
(
1C), 138.2, 129.8 (2C), 129.0 (2C), 127.3 (1C), 80.4, 54.2–53.8 EtOAc (10 mL) and washed with satd aq. NaHCO (2 × 10 mL).
3
(
2C), 52.8, 51.4 and 50.9 (1C), 47.1 and 46.9 (1C), 34.7, 30.4, The organic layer was dried over Na
2
SO
4
and concentrated
2
8.4 (3C), 21.3, 15.2 and 14.8 (1C); HRMS (ESI) calcd for in vacuo to afford the crude product, which was purified by
+
+
C H N NaO [MNa] 498.5239, found 498.5223.
flash chromatography (FC) as indicated below.
(S)-Methyl 2-((R)-4-benzoyl-3-((S)-1-(((benzyloxy)carbonyl)-
2
4
33
3
7
(
2S)-Methyl-2-(4-acetyl-3-((S)-1-((tert-butoxycarbonyl)(methyl)-
amino)-2-phenylethyl)-2,6-dioxopiperazin-1-yl)propanoate, 19. (methyl)amino)ethyl)-2-oxo-3,4-dihydropyrazin-1(2H)-yl)pro-
Prepared according to the above general procedure from 15; panoate, 23a and (S)-methyl 2-((S)-4-benzoyl-3-((S)-1-(((benzyl-
FC: ethyl acetate–n-hexane, 1 : 1.5; yield: 110 mg (77%), as an oxy)carbonyl)(methyl)amino)ethyl)-2-oxo-3,4-dihydropyrazin-1
inseparable mixture of diastereoisomers (d.e. 95%, NMR analy- (2H)-yl)propanoate, 23b. Prepared according to the above
1
sis); foam; R 0.13 (1.5 : 1 n-hexane–EtOAc); H NMR (400 MHz, general procedure from aldehyde 21 and isocyanide 3; FC:
f
CD
3
CN, 1 : 1 rotameric mixture) δ 7.37–7.19 (m, 5H), 5.50(m, ethyl acetate–n-hexane, 7 : 3; yield: 23a (24 mg, 5%), 23b
H), 5.25 (m, 1H), 5.02–4.58 (m, 2H), 4.41 (d, J = 18.6 Hz, 1H), (311 mg, 65%). 23a: thick oil; R 0.30 (7 : 3 n-hexane–EtOAc);
.66 (s, 1.5H), 3.65 (s, 1.5H), 3.04–2.77 (br, m, 2H), 2.79 (s, [α] −22.4 (c 0.5, CHCl3); H NMR (300 MHz, CDCl ) δ
1
3
3
1
f
3
0
1
D
3
H), 2.18 (s, 3H), 1.45 (d, J = 6.9 Hz, 1.5H), 1.44 (d, J = 6.9 Hz, 7.60–7.20 (m, 10H), 6.04 (br, d, J = 5.9 Hz, 1H), 5.68 (d, J = 5.9
.5H), 1.35 (br, m, 9H); C NMR (100 MHz, CD CN), δ 170.7 Hz, 1H), 5.28 (br, d, J = 9.2 Hz, 1H), 5.20–4.61 (m, 4H), 3.74 (s,
3
1
3
1
3
and 170.1 (1C), 170.0–169.7 (1C), 169.1, 167.5, 156.2 and 156.1 3H), 2.92 (s, 3H), 1.52 (d, J = 7.0 Hz, 3H), 1.24 (br, m, 3H);
C
(
1C), 137.8, 129.1, 128.9, 128.3 (2C), 126.5 and 126.4 (1C), 79.7 NMR (75 MHz, CDCl ) δ 170.9, 168.1, 162.9, 155.0, 136.7,
3
and 79.4 (1C), 56.4, 54.9 and 54.5 (1C), 52.0, 48.3, 46.7, 34.4 132.3, 130.8, 128.5–127.7 (9C), 112.3, 110.6, 67.2, 56.8, 52.6,
and 34.3 (1C), 27.9, 27.5 (3C), 20.9, 13.6 and 13.4 (1C); HRMS 52.0, 48.0, 29.7, 15.2, 14.9; HRMS (ESI) calcd for
+
+
+
+
(
4
ESI) calcd for
98.2225.
2S)-Methyl-2-(4-acetyl-3-((S)-1-((tert-butoxycarbonyl)(methyl)- NMR (400 MHz, CDCl , 1 : 1 mixture of rotamers) δ 7.61–7.40
C
24
H
33
N
3
NaO
7
[MNa] 498.2211, found
C
26
H
29
N
3
NaO
6
[MNa] 502.1949, found 502.1953. 23b: foam;
2
0
1
R
f
0.37 (7 : 3 n-hexane–EtOAc); [α]
D 3
+27.6 (c 1.0, CHCl ); H
(
3
amino)-2-phenylethyl)-2,6-dioxopiperazin-1-yl)-3-phenyl- (m, 5H), 7.39–7.27 (m, 5H), 5.96 (br, m, 0.5H), 5.88 (br, d, J =
propanoate, 20. Prepared according to the general procedure 5.4 Hz, 0.5H), 5.73 (m, 1H), 5.42–5.29 (m, 1H), 5.20–4.95 (m,
above from 16; FC: ethyl acetate–n-hexane, 1 : 1.5; yield: 3H), 4.93–4.74 (m, 1H), 3.77 (s, 1.5H), 3.74 (s, 1.5H), 3.00 (s,
1
17 mg (71%), as an inseparable mixture of diastereoisomers 1.5H), 2.94 (s, 1.5H), 1.50 (d, J = 7.1 Hz, 1.5H), 1.38 (d, J = 7.3
1
3
f 3
(d.e. 80%, NMR analysis); thick oil; R 0.39 (1.5 : 1 n-hexane– Hz, 1.5H), 1.35–1.24 (m, 3H); C NMR (100 MHz, CDCl ) δ
1
EtOAc); H NMR (400 MHz, CD CN, rotameric mixture of two 171.6 and 170.9 (1C), 168.7 and 168.6 (1C), 163.1 and 162.9
3
diastereoisomers) δ 7.36–7.07 (m, 10H), 5.56–5.40 (m, 1H), (1C), 156.6, 137.0, 134.0 and 133.9 (1C), 131.1 and 131.0 (1C),
5
.14 (d, J = 9.3 Hz, 0.9H), 5.03 (d, J = 10.5 Hz, 0.1H), 4.95–4.80 128.6–127.4 (9C), 111.9 and 111.0 (1C), 111.4 and 110.3 (1C),
(m, 0.9H), 4.65 (d, J = 18.5 Hz, 0.1H), 4.62 (d, J = 18.6 Hz, 67.3 and 67.0 (1C), 57.9 and 57.1 (1C), 52.5, 52.0 and 50.3 (1C),
0
3
.9H), 4.42 (m, 0.1H), 4.33 (d, J = 18.5 Hz, 1H), 3.71 (s, 0.3H), 50.1 and 49.0 (1C), 29.1 and 28.8 (1C), 15.9–14.4 (2C); HRMS
+
+
.66 (s, 2.7H), 3.59–3.38 (m, 1H), 3.19–3.03 (m, 1H), 2.92–2.80 (ESI) calcd for
C
26
H
29
N
3
NaO
6
[MNa] 502.1949, found
(m, 1H), 2.80–2.67 (m, 1H), 2.67 (s, 2.7H), 2.58 (s, 0.3H), 2.10 502.1961.
5002 | Org. Biomol. Chem., 2015, 13, 4993–5005
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(
S)-Methyl
methyl)amino)ethyl)-2-oxo-3,4-dihydropyrazin-1(2H)-yl)-3-phe- 9.8 Hz, 0.6H), 5.42 (d, J = 7.6 Hz, 0.4H), 5.13 (m, 1H), 5.06–4.90
nylpropanoate, 24a and (S)-methyl 2-((S)-4-benzoyl-3-((S)-1- (m, 2.6H), 4.81 (d, J = 13.0 Hz, 0.4H), 3.77 (s, 1.8H), 3.74 (s,
((benzyloxy)carbonyl)(methyl)amino)ethyl)-2-oxo-3,4-dihydro- 1.2H), 3.17–2.92 (m, 1.6H), 2.97 (s, 1.8H), 2.87 (s, 1.2H), 2.78
pyrazin-1(2H)-yl)-3-phenylpropanoate, 24b. Prepared accord- (dd, J = 13.9 and 8.1 Hz, 0.4H), 1.49 (d, J = 7.2 Hz, 1.2H), J = 7.5
2-((R)-4-benzoyl-3-((S)-1-(((benzyloxy)carbonyl)- 0.6H), 5.82–5.74 (m, 1H), 5.70 (d, J = 5.8 Hz, 0.4H), 5.51 (d, J =
(
(
1
3
ing to the above general procedure from aldehyde 21 and Hz, 1.8H); C NMR (100 MHz, CDCl
3
) δ 170.7, 169.3, 163.3,
isocyanide 4; FC: ethyl acetate–n-hexane, 3 : 7; yield: 24a 157.5 and 157.0 (1C), 138.0 and 137.8 (1C), 137.3 and 137.1
(
(
(
0
5
4
0
16 mg, 3%), 24b (223 mg, 40%). 24a: colorless oil; R 0.23 (1C), 134.5 and 134.3 (1C), 132.0 and 131.7 (1C), 129.4–127.1
f
2
0
1
7 : 3 n-hexane–EtOAc); [α]
300 MHz, CDCl ) δ 7.61–7.08 (m, 15H), 5.93 (br, d, J = 5.9 Hz, 57.4 and 54.1 (1C), 53.2 and 52.9 (1C), 51.3 and 51.0 (1C), 36.2
.7H), 5.75 (br, d, J = 5.9 Hz, 0.3H), 5.66 (d, J = 5.9 Hz, 0.7H), and 35.6 (1C), 29.9 and 29.8 (1C), 15.5 and 15.0 (1C); HRMS
D
3
−13.6 (c 0.9, CHCl ); H NMR (14C), 113.4–111.1 (2C), 67.9 and 67.5 (1C), 59.3 and 58.4 (1C),
3
+
+
.60 (d, J = 5.9 Hz, 0.3H), 5.36–4.97 (m, 4H), 4.72 (m, 0.3H), (ESI) calcd for
C
32
H
33
N
3
NaO
6
[MNa] 578.2262, found
.33 (dq, J = 10.7 and 6.8 Hz, 0.7H), 3.82 (s, 2.1H), 3.80 (s, 578.2268.
.9H), 3.48 (dd, J = 14.7 and 4.9 Hz, 0.7H), 3.43 (dd, J = 14.7
(S)-Methyl
2-((R)-4-benzoyl-3-((S)-1-(((benzyloxy)carbonyl)-
and 4.9 Hz, 0.3H), 3.17 (dd, J = 14.7 and 12.7 Hz, 0.7H), 3.04 (methyl)amino)-2-phenylethyl)-2-oxo-3,4-dihydropyrazin-1(2H)-
(
0
(
1
dd, J = 13.8 and 11.7 Hz, 0.3H), 2.92 (s, 0.9H), 2.85 (s, 2.1H), yl)-3-phenylpropanoate, 26a and (S)-methyl 2-((S)-4-benzoyl-3-
13
.73 (d, J = 6.8 Hz, 2.1H), 0.64 (d, J = 6.8 Hz, 0.9H); C NMR ((S)-1-(((benzyloxy)carbonyl)(methyl)amino)-2-phenylethyl)-2-
) δ 170.0, 168.7, 163.7 and 163.2 (1C), 155.9, oxo-3,4-dihydropyrazin-1(2H)-yl)-3-phenylpropanoate, 26b.
36.0, 135.7, 133.8, 130.8, 129.1–127.2 (14C), 111.8 and 111.3 Prepared according to the above general procedure from alde-
75 MHz, CDCl
3
(1C), 111.0 and 110.0 (1C), 67.4 and 67.0 (1C), 57.7, 56.7, 55.0, hyde 22 and isocyanide 4; FC: ethyl acetate–n-hexane, 3 : 7;
5
C
0
2.7, 34.8, 28.5, 14.6 and 14.3 (1C); HRMS (ESI) calcd for yield: 26a (56 mg, 9%), 26b (284 mg, 45%). 26a: colorless oil;
+
+
30
1
32
H
33
N
3
NaO
6
[MNa] 578.2262, found 578.2250. 24b: oil; R
f
R
f
0.22 (7 : 3 n-hexane–EtOAc); [α]
D
3
−11.1 (c 0.5, CHCl ); H
2
0
1
.27 (7 : 3 n-hexane–EtOAc); [α] +34.5 (c 1.0, CHCl ); H NMR NMR (300 MHz, CDCl , complex mixture of conformers) δ
D 3 3
3
(400 MHz, CDCl ) δ 7.56–7.18 (m, 15H), 5.90 (d, J = 5.9 Hz, 7.68–6.81 (m, 20H), 6.38 (d, J = 5.8 Hz, 0.1H), 5.94–5.80 (m,
0
0
2
.7H), 5.78 (br, d, J = 5.7 Hz, 1H), 5.72 (dd, J = 11.3 and 5.4 Hz, 0.7H), 5.79–5.72 (m. 0.2H), 5.72–5.54 (m, 1H), 5.51–5.37 (m,
.3H), 5.59 (m, 1H), 5.38 (br, d, J = 9.3 Hz, 0.7H), 5.23–5.01 (m, 0.5H), 5.31–4.65 (m, 4.5H), 3.74 (s, 0.3H), 3.70 (s, 2H), 3.67 (s,
.3H), 4.83 (br, m, 1H), 3.73 (s, 2.1H), 3.70 (s, 0.9H), 3.45 (dd, 0.7H), 3.49–3.18 (m, 1H), 3.16–2.87 (m, 3H), 2.83 (s, 0.7H),
1
3
J = 14.4 and 5.4 Hz, 0.3H), 3.40 (dd, J = 14.4 and 5.7 Hz, 0.7H), 2.80 (s, 2H), 2.72 (s, 0.3H); C NMR (75 MHz, CDCl
3
) δ
3
0
1
.02 (dd, J = 14.4 and 10.8 Hz, 1H), 2.90 (s, 2.1H), 2.86 (s, 170.2–169.6 (1C), 168.9–168.4 (1C), 163.6 and 163.1 (1C), 157.0
1
3
3
.9H), 1.25 (m, 3H); C NMR (100 MHz, CDCl ) δ 170.2, 168.6, and 156.8 (1C), 137.3–135.7 (4C), 131.8–130.9 (1C), 129.3–126.5
63.7 and 163.3 (1C), 156.6 and 156.0 (1C), 137.0 and 136.6 (19C), 112.5–106.7 (2C), 68.1–66.8 (1C), 58.0 and 57.7 (1C),
(
(
(
1C), 135.7, 133.9, 131.0, 129.3–126.9 (14C), 112.7 and 111.6 57.3–57.0 (1C), 55.8–55.3 (1C), 54.1–52.2 (2C), 38.0–37.6 (1C),
1C), 111.4 and 110.4 (1C), 67.0 and 66.9 (1C), 57.8 and 57.4 35.3–34.7 (1C), 29.4 and 29.0 (1C); HRMS (ESI) calcd for
+
+
1C), 54.6 and 54.4 (1C), 52.5, 49.9 and 49.7 (1C), 36.7, 28.9,
C
38
H
37
N
3
NaO
6
[MNa] 654.2575, found 654.2561. 26b: foam;
+
20
1
1
5.2 and 15.1 (1C); HRMS (ESI) calcd for C H N NaO
32 33 3 6
Rf 0.30 (7 : 3 n-hexane–EtOAc); [α]D −139.8 (c 1.0, CHCl ); H
NMR (400 MHz, CD CN, a mixture of two conformers 1.5 : 1) δ
3
3
+
[
MNa] 578.2262, found 578.2248.
S)-Methyl 2-((R)-4-benzoyl-3-((S)-1-(((benzyloxy)carbonyl)- 7.64–7.01 (m, 20H), 6.01 (m, 0.8H), 5.96 (d, J = 5.9 Hz, 0.6H),
methyl)amino)-2-phenylethyl)-2-oxo-3,4dihydropyrazin-1(2H)- 5.79 (d, J = 5.4 Hz, 0.6H), 5.63 (dd, J = 11.1 and 5.4 Hz, 0.4H),
(
(
yl)propanoate, 25a and (S)-methyl 2-((S)-4-benzoyl-3-((S)-1- 5.53 (dd, J = 10.8 and 5.4 Hz, 0.6H), 5.15–4.87 (m, 3.6H), 4.69
((benzyloxy)carbonyl)(methyl)amino)-2-phenylethyl)-2-oxo-3,4- (d, J = 12.9 Hz, 0.4H), 3.71 (s, 3H), 3.43 (dd, J = 14.7 and 5.4
(
dihydropyrazin-1(2H)-yl)propanoate, 25b. Prepared according Hz, 0.4H), 3.37 (dd, J = 14.7 and 5.4 Hz, 0.6H), 3.08 (dd, J =
to the above general procedure from aldehyde 22 and isocya- 14.7 and 11.1 Hz, 0.4H), 3.07 (dd, J = 14.7 and 0.81 Hz, 0.6H),
1
3
nide 3; FC: ethyl acetate–n-hexane, 1 : 1.5; yield: 25a (27 mg, 3.04–2.87 (m, 2H), 2.83 (s, 3H); C NMR (100 MHz, CD
3
CN) δ
5
%), 25b (372 mg, 67%). 25a: colorless oil; R 0.19 (1.5 : 1 170.1 and 169.9 (1C), 168.5 and 168.4 (1C), 163.6 and 163.3
f
2
0
1
n-hexane–EtOAc); [α]
D
3
−5.9 (c 0.5, CHCl ); H NMR (300 MHz, (1C), 156.6 and 155.8 (1C), 138.0 and 137.4 (1C), 136.5 and
3
CDCl ) δ 7.74–6.96 (m, 15 H), 6.47 (d, J = 6.8 Hz, 0.5H), 6.32 (d, 136.4 (2C), 134.2 and 133.8 (1C), 131.1 and 130.9 (1C),
J = 6.8 Hz, 0.5H), 6.09–6.01 (m, 0.5H), 5.93–5.84 (m, 0.5H), 129.1–126.4 (19C), 112.0–111.5 (2C), 66.5 and 66.3 (1C), 57.2
5
1
.36–4.83 (m, 4.5H), 4.49 (m, 0.5H), 3.75 (s, 1.5H), 3.70 (s, and 57.1 (1C), 56.0, 55.1–54.8 (2C), 52.2 and 52.1 (1C), 36.1
13
.5H), 3.18–2.69 (m, 5H), 1.32 (d, J = 7.8 Hz, 3H); C NMR and 35.9 (1C), 34.9 and 34.6 (1C), 29.0 and 28.8 (1C); HRMS
+
+
(75 MHz, CDCl ) δ 173.0, 167.4, 162.8, 160.2 and 159.6 (1C), (ESI) calcd for C H N NaO
[MNa] 654.2575, found
3
38 37
3
6
1
37.2, 136.2, 133.0, 131.8, 129.4–127.1 (14C), 112.1–112.3 (2C), 654.2558.
6
2
5
8.1 and 66.8 (1C), 53.4, 52.8, 52.5, 48.3, 37.9 and 37.6 (1C),
+
+
Computational studies
Conformational analysis of compound 9. MMFF and DFT
H NMR calculations were run with Spartan’10 (Wavefunction, Inc.,
9.1, 18.2; HRMS (ESI) calcd for C H N NaO
[MNa]
0.35 (1.5 : 1
3
2
33
3
6
78.2262, found 578.2270. 25b: pale yellow oil; R
f
2
0
1
n-hexane–EtOAc); [α]
D
−72.1 (c 0.5, CHCl
3
);
(
400 MHz, CDCl ) δ 7.61–7.11 (m, 15H), 5.91 (br, d, J = 5.6 Hz, Irvine, CA, 2010), with standard parameters and convergence
3
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2
7
criteria. TDDFT calculations were run with Gaussian’09, with (40, 20, 10, 5, and 2.5 µM) dissolved in dimethyl sulfoxide
default grids and convergence criteria. Conformational (DMSO). The corresponding DMSO vehicles were also applied
searches were run with the Monte Carlo algorithm to Huh7 and Mahlavu cells as negative controls. DMSO con-
implemented in Spartan’10 using Merck molecular force field centrations were always below 0.01%. After 72 h, cells were
(
MMFF) in vacuo. All structures thus obtained were optimized washed with 1×PBS (CaCl -, MgCl -free) (Gibco, Invitrogen),
2 2
with the DFT method using the B3LYP functional and the and then fixed with cold 10% (v/v) trichloroacetic acid
-31G(d) basis set in vacuo. TDDFT calculations were run using (MERCK). Microplates were left for 1 h at 4 °C, then washed
6
the CAM-B3LYP functional and the SVP basis set, including 36 with water and left to dry. The plates were then stained with
excited states (roots). On some representative structures, we 100 μl of 0.4% sulphorhodamine B (SRB) (Sigma Aldrich) in a
verified the consistency with the results obtained with the 1% acetic acid solution for 10 min. The unbound SRB was
larger TZVP basis set. CD spectra were generated using the washed with 1% acetic acid. SRB was then solubilized in 200 μl
2
9
program SpecDis by applying a Gaussian band shape with of 10 mM Tris-base solution. The absorbance was read at
.3 eV exponential half-width, from dipole-length rotational 515 nm. The experiment was performed in duplicate and the
strengths. The difference with dipole-velocity values was found absorbance values were normalized to DMSO controls and Tz
0
to be minimal for all relevant transitions.
values. Standard deviations were less than 10%.
Computational studies on models a, b and c. The starting
geometries of compounds a, b and c (Fig. 4), created by Gauss-
27
View, were energy minimized by the conjugate gradient
Notes and references
2
7
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5
1
2. GAFF force fields were used for the parameterization of
the molecules, modeled as neutral compounds in an implicit
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2
6
water). With a time step of 2 fs, each peptidomimetic was
heated to 300 K, 700 K and then to 1000 K over 20 ps. After an
equilibration phase of 2 ns, each model were frozen to 700 K
and then to 300 K over 20 ps. The production run of the MD
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tories saved every 10 ps. The resulting structures in the trajec-
3
0
tories were visually analyzed by VMD. In this stage, the
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β
percentage distribution (Fig. 4). C –C distances were
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NCI-60 Sulforhodamine B (SRB) assay. Huh7 and Mahlavu
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