The diketopiperazine-fused tetrahydro-β-carboline scaffold as a model peptidomimetic with an unusual α-turn secondary structure

Article abstract of DOI:10.3762/bjoc.9.17

Aiming at restricting the conformational freedom of tryptophan-containing peptide ligands, we designed a THBC (tetrahydro-β- carboline)-DKP (diketopiperazine)-based peptidomimetic scaffold capable of arranging in an unusual α-turn conformation. The synthesis is based on a diastereoselective Pictet-Spengler condensation to give the THBC core, followed by an intramolecular lactamization to complete the tetracyclic THBC-DKP fused ring system. The presence of conformers bearing the intramolecular thirteen-membered hydrogen bond that characterizes the α-turn structure is confirmed by 1H NMR conformational studies. To the best of our knowledge, this scaffold represents one of the rare examples of a designed constrained α-turn mimic.

Full text of DOI:10.3762/bjoc.9.17

The diketopiperazine-fused tetrahydro-β-carboline
scaffold as a model peptidomimetic with an unusual
α-turn secondary structure
Francesco Airaghi1, Andrea Fiorati2, Giordano Lesma1, Manuele Musolino1,
Alessandro Sacchetti*2 and Alessandra Silvani*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 147–154.
1Dipartimento di Chimica, Università degli Studi di Milano, via Golgi
19, 20133 Milano, Italy and 2Dipartimento di Chimica, Materiali ed
Ingegneria Chimica ‘Giulio Natta’, Politecnico di Milano, piazza
Leonardo da Vinci 32, 20132 Milano, Italy
doi:10.3762/bjoc.9.17
Received: 07 November 2012
Accepted: 20 December 2012
Published: 22 January 2013
Email:
Alessandro Sacchetti* - alessandro.sacchetti@polimi.it;
Alessandra Silvani* - alessandra.silvani@unimi.it
Associate Editor: N. Sewald
© 2013 Airaghi et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
α-turn; conformational analysis; diketopiperazine; peptidomimetics;
tetrahydro-β-carboline
Abstract
Aiming at restricting the conformational freedom of tryptophan-containing peptide ligands, we designed a THBC (tetrahydro-β-
carboline)-DKP (diketopiperazine)-based peptidomimetic scaffold capable of arranging in an unusual α-turn conformation. The
synthesis is based on a diastereoselective Pictet–Spengler condensation to give the THBC core, followed by an intramolecular
lactamization to complete the tetracyclic THBC-DKP fused ring system. The presence of conformers bearing the intramolecular
thirteen-membered hydrogen bond that characterizes the α-turn structure is confirmed by 1H NMR conformational studies. To the
best of our knowledge, this scaffold represents one of the rare examples of a designed constrained α-turn mimic.
Introduction
From a long time, the alkaloids containing the 1,2,3,4- compounds are heterocyclic scaffolds structurally similar to
tetrahydro-β-carboline (THBC) skeleton have represented peptides. They have attracted attention in recent years because
important lead structures in view of their wide range of bio- of their broad biological activities [11,12] and therapeutic appli-
logical activities [1], mainly due to their interaction with the cations, ranging from antibiotics [13] to anticancer agents [14].
central nervous system [2-7]. Moreover, recently some tetra- Moreover, the DKP moiety has been exploited as a
cyclic β-carbolines have been described to act as selective peptidomimetic scaffold [15-17]. Structural unification of
inhibitors in the anticancer field [8,9], or to be endowed with THBC and DKP pharmacophores has led to new classes of bio-
antimalarial properties [10]. 2,5-Diketopiperazine (DKP)-based logically active tetracyclic compounds, both naturally occur-
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Beilstein J. Org. Chem. 2013, 9, 147–154.
ring and synthetically made [18]. Highly complex natural prod- Even if a very large majority of α-turn segments form a part of
ucts displaying the fused, tetracyclic THBC-DKP ring system regular α-helices, isolated α-turns have been reported [35],
were recently isolated from the fungus Aspergillus fumigatus which are stabilized by a 5→1 hydrogen bond between the car-
[19] and have been shown to exhibit significant cell growth bonyl oxygen at position i and the amide at position i+4.
inhibitory activities against various cell lines [20]. Specifically
designed THBC-DKP-based compounds have received consid- Quite recently the presence of α-turns in constrained peptides
erable attention over the past few years for their valuable bio- has been associated with various relevant biological activities,
logical activities, ranging from the inhibition of the cyclic highlighting potential applications in the field of bacteriolytic
guanosine monophosphate type 5 specific phosphodiesterase [36], antiviral [37] and anti-HIV compounds [38]. Other exam-
(PDE 5) for the treatment of erectile dysfunction (Tadalafil) ples of α-turn conformations are described in synthetic
[21], and the inhibition of plasmodial PDE activity for antima- peptidomimetics [36,39-42].
larial drugs [22,23], to topoisomerase II inhibition [24], and oral
antithrombotic properties [25] (Figure 1).
Despite the growing interest in this kind of reverse turn and the
need for all kinds of conformationally constrained mimics as
In our ongoing program of design of pharmacophore-based tools for medicinal chemistry, the development of constrained
combinatorial libraries [26] and identification of new α-turn mimetics has received little attention until now. We
peptidomimetic scaffolds of potential interest in drug discovery report here the synthesis and conformational evaluation of the
[27-30], we evaluated the THBC-DKP-based scaffold as a THBC-DKP-based peptidomimetic 1a (Figure 2), for which
potential suitable motif for the creation of unusual reverse-turn molecular modeling allowed us to envisage a 13-membered
nucleators.
hydrogen-bond-stabilized α-turn conformation.
Reverse turns are structural motifs commonly found in bioac- Results and Discussion
tive peptides, which, besides being fundamental in protein In order to investigate the presence of a preferred conformation
folding, play a central role as molecular-recognition elements able to mimic an ordered protein secondary structure, a
[31-34]. In addition to the most frequently occurring β and computer-aided conformational analysis was performed on 1a
γ-turns, reversal of the polypeptide chain direction in globular (6S,12aS-configuration, IUPAC atom numbering as in Figure 2)
proteins can also occur thanks to less common substructures, for and diastereoisomeric 1b (6R, 12aS). Compounds 1a and 1b
example involving five amino acids residues, such as the case were submitted to an unconstrained Monte Carlo (MC) con-
of the α-turns.
formational search combined with Molecular Mechanics (MM)
Figure 1: THBC-DKP-based natural and synthetically made compounds.
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Beilstein J. Org. Chem. 2013, 9, 147–154.
1a). Inspection of the global minima showed the α-turn con-
formation only for compound 1a. For this diastereoisomer the
first four low-energy conformers all adopt the α-turn con-
formation (presence of the 5→1 H-bond and average
dα = 5.1 Å): according to a Boltzmann distribution analysis,
these conformers take into account 94.5% of all the conformers
obtained by the MC/MM analysis. On the other hand, for com-
pound 1b, the lowest energy conformers all have the 5→3
H-bond (classical γ-turn), with the only α-turn conformation
lying 5.9 kcal/mol above the global minimum. In Figure 4 the
low-energy conformers of 1a and 1b are represented. These
pictures highlight well the crucial role of the C6-configuration
Figure 2: THBC-DKP-based peptidomimetic 1a.
minimization (see Table 1 for results). As a main indication of a in favoring either an α-turn conformation (1a) or a γ-turn con-
stable secondary structure, the presence of intramolecular formation (1b).
hydrogen bonds was first evaluated. Two H-bonds have been
identified, i.e., a 7-membered-ring H-bond (H-bond A, Figure 3)
between the N5H hydrogen and the C3 carbonyl and a
13-membered-ring H-bond (H-bond B) between the N5H
hydrogen and the C1 carbonyl, here represented by the Cbz car-
bonyl. To assess the presence of turn conformations we also
measured the interatomic distance dα between the terminal Cα5
atom and the benzyl oxygen of the Cbz (which emulates the
Cα1 atom), assuming a value dα < 7 Å as a probe of a generic
reverse turn. Results are reported as the number of conformers
that meet the geometric requirements.
Figure 4: Perspective view of the low-energy conformers of 1a,b.
Hydrogen atoms are omitted for clarity.
Table 1: MC/EM conformational analysis for peptidomimetics 1a and
1b. The + symbol indicates the presence in the global minimum.
Finally, having ascertained by means of further calculations the
Conf. within
6 kcal/mol
H-bond A
H-bond B
dα < 7 Å
irrelevance of the C3 configuration on the expected secondary
structure, we fixed it as 3S. Being interested in unusual reverse
turns, we then pursued the synthesis of peptidomimetic 1a
(Scheme 1). Starting from L-tryptophan methyl ester and
N-Cbz-aminoacetaldehyde dimethyl acetal [43], tetrahydro-β-
carboline 2 was obtained in good yield and high diastereoselec-
tivity (dr 70% from 1H NMR) by means of Pictet–Spengler
reaction [44] and subsequent chromatographic separation.
1a
1b
35
33
17
4 +
1
4 +
1
22 +
The 6S,12aS-configuration of the prevailing diastereoisomer
was easily ascertained by application of the protocol of
Ungemach et al. [45] on the 13C NMR spectrum of compound 2
and, conclusively, by the observation of an intense NOE contact
between H-6 and H-12a in the 2D NOESY spectrum. Alkyl-
ation of L-alanine benzyl ester with ethyl bromoacetate afforded
amine 3 [46], on which N-Fmoc protection and subsequent care-
fully conducted hydrogenolysis of the benzyl ester were
performed [47], to give acid 4 in acceptable overall yield.
Figure 3: Geometric parameters for mimics 1a,b.
The most frequently observed is the 5→3 H-bond (H-bond A),
which is related to a classical γ-turn around these residues, and Condensation of acid 4 with secondary amine 2 proved to be
a 5→1 H-bond (H-bond B), which can be identified as an α-turn troublesome under a wide range of conditions, probably due to
(this conformation is present in four conformers for compound the severe steric hindrance of both the amine and acid coupling
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Beilstein J. Org. Chem. 2013, 9, 147–154.
Scheme 1: Synthesis of the THBC-DKP-based peptidomimetic 1a.
partners. At the end, acceptable yields could be obtained via is, in the absence of any noticeable intermolecular aggregation.
formation of the chloride intermediate [48], by reaction of acid While both NHMe and NHCbz chemical-shift values are low
4 with thionyl chloride, and subsequent coupling with 2 in (6.78 ppm for NHMe and 5.53 ppm for NHCbz), a significant
CH2Cl2 and 2,6-lutidine to give 5. The formation of the fused, difference could be appreciated between their temperature coef-
tetracyclic THBC-DKP ring system was then easily achieved by ficients, ranging from 7.0 ppb K−1 (in absolute value) for the
removal of the N-Fmoc protecting group [49] and spontaneous NHMe signal to 2.3 ppb K−1 (in absolute value) for the NHCbz
lactamization to give the diketopiperazine ring of 6. The signal. According to the literature [50], these data could be
obtained compound 6 represents a valuable peptidomimetic, attributed to a situation of equilibrium between hydrogen-
whose potential is also related to the possibility of further bonded and non-hydrogen-bonded states for the NHMe amide
derivatization with desired pharmacophoric groups, on both the proton and a completely non-hydrogen-bonded state for the
terminal acid and amine functional groups, for the development NHCbz amide proton. In addition, a supplementary indication
of conformationally constrained tryptophan-containing peptide of the different hydrogen-bonding state for the two NH protons
ligands.
was obtained from DMSO titration studies in CDCl3 [51], indi-
cating that the chemical shift of the NHMe has a minor varia-
To investigate the actual secondary structure of the THBC-DKP tion (0.24 ppm) upon addition of up to 30% of the competitive
scaffold also in solution, compound 6 was converted into the solvent DMSO, with respect to the chemical shift of NHCbz,
N-methyl carboxyamide derivative 1a, by a two-step procedure i.e., varying by 0.82 ppm.
(0.5 M LiOH, 0 °C, then MeNH2, TBTU, DIPEA), which was
carefully conducted in order to avoid the easy epimerization of Taking into account the suggestions from the molecular
the C3 and C12a stereocenters. Spin-system identification and modeling and these experimental results, for 1a we can presume
assignment of individual resonances for peptidomimetic model the presence in solution of conformers bearing a 13-membered
1a was straightforward with 1H COSY. The study of the con- intramolecular hydrogen bond involving the NHMe proton and
formational behavior was conducted in CDCl3, to identify the Cbz carbonyl group, as visualized in the perspective view of
possible intramolecular hydrogen bonding.
the low energy conformer of 1a.
The involvement of the NH amide protons in such bonding was Conclusion
first estimated from evaluation of their chemical shift value (δ) In conclusion, we realized the synthesis of a new constrained
and of the temperature coefficients Δδ/ΔT (between 263 and THBC-DKP-based scaffold able to mimic an α-turn. It was
328 K). All data were measured at 2.0 mM concentration, that designed with the aid of computational tools, which highlight
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Beilstein J. Org. Chem. 2013, 9, 147–154.
the relative cis arrangement of the substituents on the THBC Characterization of 2. Oil; [α]D −44.4 (c 1, CHCl3); 1H NMR
piperidine ring as being a crucial requirement in order to obtain (CDCl3, 400 MHz) δ 8.96 (1H, br s), 7.55–7.10 (9H, m), 6.02
the correct geometry for mimicry. Following these studies, the (1H, br, dd, NH-Cbz), 5.03 (2H, s), 4.40 (1H, br s), 3.90–3.60
desired isomer 1a of the THBC-DKP based peptidomimetic was (6H, m), 3.18 (1H, dd, J = 15.0, 1.6 Hz), 2.88 (1H, dd, J = 15.0,
synthesized. 1H NMR conformational studies confirmed the 11.2 Hz), 2.77 (1H, br s); 13C NMR (CDCl3, 100 MHz) δ
presence of the intramolecular thirteen-membered hydrogen 173.5, 157.3, 136.5, 136.3, 132.3, 128.5–127.8 (5C), 127.0,
bond that characterizes the α-turn conformation, even if a situa- 122.0, 119.5, 118.0, 111.3, 109.2, 66.9, 56.3, 53.6, 52.3, 44.3,
tion of equilibrium between hydrogen-bonded and non- 25.5; IR (cm−1): 3018, 1709, 1514, 1362, 1267, 1228;
hydrogen-bonded states can be observed. Nevertheless, this HRMS–EI (m/z): [M+] calcd for C22H23N3O4, 393.1689; found,
scaffold represents one of the rare examples of a designed 393;1702.
constrained α-turn mimic. Its application to the synthesis of bio-
logically valuable peptides embodying an α-turn core is (S)-Benzyl 2-((2-ethoxy-2-oxoethyl)amino)propanoate (3):
currently under way.
Under a nitrogen atmosphere L-alanine-benzyl ester (500 mg,
2.79 mmol, 1 equiv) was dissolved in CH3CN (6 mL, 0.47 M),
then K2CO3 (3.85 g, 27.90 mmol, 10 equiv) was added. Ethyl
Experimental
General information: All commercial materials (Aldrich, bromoacetate (466 mg, 2.79 mmol, 310 µL, 1 equiv) was added
Fluka) were used without further purification. All solvents were to the suspension, and the mixture was reacted overnight at
reagent grade or HPLC grade. All reactions were carried out room temperature. The solvent was evaporated under reduced
under a nitrogen atmosphere unless otherwise noted. All reac- pressure, and the crude material was dissolved in AcOEt
tions were monitored by thin-layer chromatography (TLC) on (20 mL). The solution was extracted with an aqueous solution
precoated silica gel 60 F254; spots were visualized with UV of H3PO4 5% (3 × 25 mL), and the reunited aqueous layers
light or by treatment with 1% aqueous KMnO4 solution. Prod- were basified with Na2CO3 until pH 9 and extracted repeatedly
ucts were purified by flash chromatography on silica gel 60 with AcOEt. The reunited organic phases were dried with
(230–400 mesh). 1H NMR spectra and 13C NMR spectra were Na2SO4, filtered and evaporated under reduced pressure, to give
recorded on 300, 400 and 500 MHz spectrometers. Chemical compound 3 (503 mg, 67% yield), as an oil. [α]D −17.11 (c 1,
shifts are reported in parts per million relative to the residual CHCl3); 1H NMR (CDCl3, 300 MHz) δ 7.4–7.28 (5H, m),
proton resonance of the solvent. 13C NMR spectra were 5.2–5.1 (2H, m), 4.15 (2H, q, J = 8.6 Hz), 3.53 (1H, q,
recorded by using the APT pulse sequence. Multiplicities in J = 9.6 Hz), 3.49 (1H, d, J = 17.3 Hz), 3.39 (1H, d,
1H NMR are reported as follows: s = singlet, d = doublet, J = 17.3 Hz), 2.37 (1H, br s), 1.38 (3H, d, J = 9.6 Hz), 1.25 (3H,
t = triplet, m = multiplet, br s = broad singlet. High-resolution t, J = 8.6 Hz); 13C NMR (CDCl3, 75.4 MHz) δ 174.3, 171.4,
MS spectra were recorded with a FT-ICR (Fourier Transform 135.6, 128.6–128.2 (5C), 66.7, 61.0, 55.9, 48.7, 18.6, 14.2;
Ion Cyclotron Resonance) instrument, equipped with an ESI HRMS–EI (m/z): [M+] calcd for C14H19NO4, 265.1314; found,
source, or a standard MS instrument, equipped with an EI 265.1328.
source. Yields refer to chromatographically and spectroscopi-
cally pure compounds.
(S)-2-((9H-Fluoren-9-ylmethoxycarbonyl)(2-ethoxy-2-
oxoethyl)amino)propanoic acid (4): Under a nitrogen atmos-
(1S,3S)-Methyl 1-(benzyloxycarbonylaminomethyl)-2,3,4,9- phere, compound 3 (1.49 g, 5.62 mmol, 1 equiv) and DIPEA
tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (2): Under (871 mg, 6.74 mmol, 1.17 mL, 1.2 equiv) were dissolved in
a nitrogen atmosphere, L-tryptophan methyl ester (755 mg, CH2Cl2 (40 mL, 0.14 M), then FmocCl (1.48 g, 5.73 mmol,
3.46 mmol, 1.0 equiv) was dissolved in CH2Cl2 (30 mL, 1.02 equiv) was added. The mixture was stirred at room
0.12 M), then N-Cbz-aminoacetaldehyde dimethyl acetal temperature overnight. Then CH2Cl2 (10 mL) was added, the
(912 mg, 3.81 mmol, 1.1 equiv) was added. The solution was solution was washed with HCl 0.5 M (3 × 30 mL), and the
cooled to −30 °C, then TFA was added (1.97 mg, 1.3 mL, organic layer was dried with Na2SO4, filtered and evaporated
17.3 mmol, 5 equiv). The mixture was kept for 2 h at this under reduced pressure. The crude was dissolved in MeOH
temperature then reacted overnight at room temperature. The (100 mL, 0.045 M), and then, under a nitrogen atmosphere,
solution was diluted with AcOEt and washed three times with a Pd/C 5% w/w (150 mg) was added. The resulting suspension
saturated solution of NaHCO3 (3 × 15 mL), dried over Na2SO4, was stirred for 3 h under a hydrogen atmosphere at room
filtered and evaporated under reduced pressure. The crude mix- temperature. The reaction was accurately monitored by TLC in
ture was purified by chromatographic column (n-hexane/AcOEt order to prevent hydrogenolysis of the Fmoc group. The suspen-
6:4), and 808 mg of (1S,3S)-2 and 143 mg of the (1R,3S)-dia- sion was filtered on a layer of celite, the solution was evapo-
stereoisomer were obtained (70% overall yield, 70% dr).
rated under reduced pressure, and the crude was dissolved in
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Beilstein J. Org. Chem. 2013, 9, 147–154.
AcOEt and then extracted three times with a saturated solution Compound 5 (300 mg, 0.39 mmol) was dissolved in THF
of NaHCO3. The reunited aqueous phases were acidified with (13 mL, 0.03 M), and then piperidine was added (2.6 mL). The
HCl (0.5 M) and extracted with AcOEt (3 × 35 mL), then dried solution was reacted overnight, then HCl (0.5 M) was added,
with Na2SO4, filtered and evaporated under reduced pressure, and the mixture was extracted with AcOEt (3 × 30 mL). The
to give pure 4 (1.76 g, 79% overall yield) as an oil. [α]D −9.42 reunited organic layers were dried with Na2SO4, filtered and
(c 1, MeOH); 1H NMR (DMSO, 300 MHz, 95 °C) δ 7.88 (2H, evaporated under reduced pressure. The crude material was
d, J = 7.5 Hz), 7.64 (2H, d, J = 7.4 Hz), 7.43 (2H, t, J = 7.3 Hz), purified by column chromatography (AcOEt/n-hexane, 4:6 to
7.34 (2H, t, J = 7.3 Hz), 4.58 (1H, q, J = 7.3 Hz), 4.38 (2H, d, 6:4), to give 6 (165 mg, 81% yield) as an oil. [α]D −59.5 (c 1,
J = 6.3 Hz), 4.26 (1H, t, J = 6.3 Hz), 4.12 (2H, q, J = 7.1 Hz), CHCl3); 1H NMR (CDCl3, 400 MHz, major rotamer) δ 9.27
3.98 (2H, AB system, J = 18.0 Hz), 1.37 (3H, d, J = 7.3 Hz), (1H, br s), 7.55–7.10 (9H, m), 5.91 (1H, br s), 5.15–5.09
1.21 (3H, t, J = 7.1 Hz); 13C NMR (CDCl3, 75.4 MHz, mixture (3H, m), 4.50 (1H, d, J = 17.2 Hz), 4.26 (4H, m), 4.05 (1H, m),
of two rotamers) δ 175.4 and 174.5, 171.8 and 170.7, 155.7 and 3.97 (1H, d, J = 17.5 Hz), 3.68 (1H, m), 3.57 (1H, br d,
155.6, 143.7, 143.6, 141.2 (2C), 127.7 (2C), 127.1 (2C), 124.9 J = 15.6 Hz), 3.07 (1H, dd, J = 15.6, 11.8 Hz), 1.58 (3H, d,
(2C), 120.0 (2C), 68.6 and 68.2, 62.2 and 61.8, 55.7 and 55.5, J = 12.8 Hz), 1.31 (3H, t, J = 7.2 Hz); 13C NMR (CDCl3,
47.6 and 47.1, 47.0, 15.5 and 14.9, 14.1; IR (cm−1): 2954, 1750, 75.4 MHz) δ 168.9, 168.5, 167.3, 157.6, 136.5, 136.3, 130.7,
1709, 1467, 1451, 1325, 1204; HRMS–EI (m/z): [M+] calcd for 128.6, 128.2, 128.0, 126.2, 122.5, 119.9, 119.8, 118.3, 111.7,
C22H23NO6, 397.1525; found, 397.1541.
107.7, 67.1, 61.8, 57.6, 57.2, 56.3, 45.0, 44.7, 31.6, 25.2, 17.9,
14.2; IR (cm−1): 3016, 1742, 1667, 1506, 1455, 1326, 1222.
(1S,3S)-Methyl 2-((S)-2-((9H-fluoren-9-ylmethoxy- HRMS–EI (m/z): [M+] calcd for C28H30N4O6, 518.2165; found,
carbonyl)(2-ethoxy-2-oxoethyl)amino)propanoyl)-1-(ben- 518.2178.
zyloxycarbonylaminomethyl)-2,3,4,9-tetrahydro-1H-
pyrido[3,4-b]indole-3-carboxylate (5): Under a nitrogen Benzyl (((3S,6S,12aS)-3-methyl-2-(2-(methylamino)-2-
atmosphere, compound 4 (302 mg, 0.76 mmol, 1 equiv) was oxoethyl)-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyr-
dissolved in CH2Cl2 (4 mL, 0.19 M), the solution was cooled to azino[1',2':1,6]pyrido[3,4-b]indol-6-yl)methyl)carbamate
0 °C, and then SOCl2 (904 mg, 7.60 mmol, 550 µL, 10 equiv) (1a): Compound 6 (165 mg, 0.32 mmol, 518 g/mol) was
was added. The mixture was stirred at room temperature for 5 h dissolved in EtOH (3 mL). Aqueous LiOH (0.5 M, 2.54 mmol,
then evaporated under reduced pressure. The obtained crude 4.6 mL) was added and the reaction was stirred at 0 °C for 1 h.
material was dissolved in CH2Cl2 (3 mL) and slowly added to a After then, the solution was acidified with HCl (1 N, 8 mL),
solution of 2 (299 mg, 0.76 mmol, 1 equiv) and 2,6-lutidine extracted with AcOEt (3 × 20 mL), dried, filtered and concen-
(110 mg, 1.03 mmol, 120 μL, 1.35 equiv) in CH2Cl2 (5 mL), trated. The crude material was suspended in CH2Cl2 (10 mL)
cooled at 0 °C under nitrogen atmosphere. The mixture was under N2, DIPEA (208 µL, 1.20 mmol, 129 g/mol) and TBTU
stirred at room temperature overnight, then CH2Cl2 (10 mL) (384 mg, 1.20 mmol, 321 g/mol) were added. After stirring for
was added, the organic layer was washed with HCl (0.5 M, 30 min, MeNH2 (2.0 M THF solution, 1.20 mmol, 601 µL) was
5 mL), saturated aq NaHCO3 (5 mL) and brine (5 mL). The added. The reaction was stirred for 24 h under N2, and then it
organic phase was dried with Na2SO4, filtered and evaporated was poured into water (20 mL) and extracted with AcOEt
under reduced pressure. The crude material was purified by (3 × 20 mL), dried, filtered and concentrated. Purification by
column chromatography (n-hexane/AcOEt 6:4), affording 5 flash chromatography (AcOEt) afforded product 1a (110 mg,
(321 mg, 55% yield) as an oil. [α]D +17.87 (c 1 CHCl3), 68% overall yield) as an oil. [α]D −98.3 (c 0.75, MeOH);
1H NMR (CDCl3, 300 MHz, major rotamer) δ 9.87 (1H, br s), 1H NMR (500 MHz, CD3CN, major rotamer) δ 9.41 (1H, br s),
7.88–7.00 (17H, m), 6.07 (1H, br s), 5.45 (1H, br q), 5.32 (1H, 7.55 (1H, d, J = 7.6 Hz), 7.38 (1H, d, J = 7.8 Hz), 7.34–7.23
br s), 5.23 (2H, br s), 4.71–4.32 (2H, m), 4.20–4.10 (5H, m), (5H, m), 7.13 (1H, ddd, J = 7.8, 7.6 and 0.9 Hz), 7.06 (1H, dt,
4.08–3.74 (3H, m), 3.74–3.08 (5H, m), 1.48 (3H, d, J = 7.5 Hz), J = 7.6 and 0.9 Hz), 6.95 (1H, br q, J = 4.7 Hz), 5.83 (1H, br q,
1.25 (3H, d, J = 7.3 Hz); 13C NMR (CDCl3, 100 MHz, major J = 8.2 and 5.1 Hz), 5.43 (1H, br t, J = 3.4 Hz), 4.90 (1H, d,
rotamer) δ 171.1, 169.3, 169.1, 155.9, 154.2, 143.9, 141.2, J = 12.3 Hz), 4.87 (1H, d, J = 12.3 Hz), 4.26 (1H, d,
136.4, 130.6, 128.5–111.5 (22C), 68.8, 67.1, 61.4, 56.3, 54.8, J = 17.0 Hz), 4.16 (1H, q, J = 7.0 Hz), 4.14 (1H, dd, J = 11.4
52.1, 51.0, 47.1, 47.0, 46.6, 24.4, 15.3, 14.2; HRMS–EI (m/z): and 5.5 Hz), 3.98 (1H, ddd, J = 14.2, 8.2 and 3.8 Hz), 3.94 (1H,
[M+] calcd for C44H44N4O9, 772.3108; found, 772.3095.
d, J = 17.0 Hz), 3.43 (1H, dd, J = 15.6 and 5.5 Hz),
3.26 (1H, ddd, J = 14.2, 5.1 and 3.2 Hz), 3.00 (1H, dd,
Ethyl 2-((3S,6S,12aS)-6-(benzyloxycarbonylaminomethyl)-3- J = 15.6 and 11.4 Hz), 2.69 (3H, d, J = 4.7 Hz), 1.48
methyl-1,4-dioxo-3,4,12,12a-tetrahydropyrazino- (3H, d, J = 7.0 Hz); 13C NMR (125.8 MHz, CD3CN) δ 170.4,
[1',2':1,6]pyrido[3,4-b]indol-2(1H,6H,7H)-yl)acetate (6): 170.0, 169.8, 157.8, 137.5, 135.2, 132.0, 129.0 (2C),
152

Beilstein J. Org. Chem. 2013, 9, 147–154.
11.Wang, S.; Golec, J.; Miller, W.; Milutinovic, S.; Folkes, A.; Williams, S.;
Brooks, T.; Hardman, K.; Charlton, P.; Wren, S.; Spencer, J.
Bioorg. Med. Chem. Lett. 2002, 12, 2367–2370.
128.5, 128.3 (2C), 126.9, 122.2, 119.8, 118.7, 112.0, 108.1,
66.7, 55.4, 55.3, 53.5, 46.7, 44.7, 22.7, 25.9, 14.5;
HRMS–EI (m/z): [M+] calcd for C27H29N5O5, 503.2169; found,
503.2186.
doi:10.1016/S0960-894X(02)00389-X
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Computational details
13.Fdhila, F.; Vazquez, V.; Sanchez, J. L.; Riguera, R. J. Nat. Prod. 2003,
66, 1299–1301. doi:10.1021/np030233e
An unconstrained Monte Carlo (MC) conformational search
combined with Molecular Mechanics (MM) minimization was
performed by using the software Spartan’08 [52]. Images were
elaborated with the software PyMol [53]. For each compound
972 conformers were generated, and after removal of dupli-
cated minima, only conformations within 6 kcal/mol of the
global minimum were considered. Thirty-five and thirty-three
conformers were kept for compounds 1a and 1b, respectively.
Inspection of the virtual torsion angles of the amide backbone
revealed the preference for an inverse γ-turn of the low-energy
conformers of 1b (φ = −80.1° and ψ = 79.6° on average, based
on the conformers accounting 93% of all the conformers from a
Boltzmann distribution analysis). For compound 1a the aver-
ages of the torsion angles were φ (Cα2) = −81.5°,
ψ (Cα2) = 64.5°, φ (Cα3) = 146.3°, ψ (Cα3) = 36.9°,
φ (Cα4) = 120.5°, and ψ (Cα4) = −29.3°. According to the classi-
fication of α-turns in protein structures [32], compound 1a is an
α-turn mimetic with a B-αL-X designation.
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