Formation of a Pseudo-β-hairpin motif utilizing the Ant-pro reverse turn: Consequences of stereochemical reordering

Article abstract of DOI:10.1039/c3ob42016g

Herein, we report a special case of pseudo-β-hairpin formation by tetrapetide sequences featuring a two-membered Ant-Pro dipeptide motif (Ant = anthranilic acid and Pro = proline) at the loop region. These folded structures uniquely feature the presence of C9- and C17-H-bonding patterns at reverse turn and interstrand regions, respectively. Their hairpin nucleation and folding propensities have been expounded using solution and solid state studies of distinct stereochemically altered sequences.

Full text of DOI:10.1039/c3ob42016g

Organic &
Biomolecular Chemistry
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Formation of a pseudo-β-hairpin motif utilizing
the Ant–Pro reverse turn: consequences of
stereochemical reordering†
Cite this: Org. Biomol. Chem., 2014,
12, 774
Roshna V. Nair,a Amol S. Kotmale,b Snehal A. Dhokale,b Rupesh L. Gawade,c
Vedavadi G. Puranik,c Pattuparambil R. Rajamohananb and Gangadhar J. Sanjayan*a
Herein, we report a special case of pseudo-β-hairpin formation by tetrapetide sequences featuring a two-
membered Ant–Pro dipeptide motif (Ant = anthranilic acid and Pro = proline) at the loop region. These
folded structures uniquely feature the presence of C9- and C17-H-bonding patterns at reverse turn and
interstrand regions, respectively. Their hairpin nucleation and folding propensities have been expounded
using solution and solid state studies of distinct stereochemically altered sequences.
Received 8th October 2013,
Accepted 18th November 2013
DOI: 10.1039/c3ob42016g
www.rsc.org/obc
β-Hairpin is one of the smallest secondary structural
folding motifs that causes augmentation of the β-sheet second-
Introduction
ary structure in proteins.9 Further, β-hairpins are amongst the
most preferred motifs/candidates for ‘protein epitope mimetic’
design owing to their participation in various molecular recog-
nition events.10 Development of such mimics is expected to
further widen the scope of peptides in practical utility.
Incorporation of diverse synthetic secondary structural
elements into a functional bioactive peptide core has increas-
ingly meliorated the understanding of the interactions of
small molecules with biological targets such as enzymes/recep-
tors, etc.1 This aspect of peptidomimetics has led to rapid pro-
gress in peptide-based catalysis and therapeutics.2 In this
connection, our group has been keenly involved in developing
hybrid aliphatic–aromatic amino acid conjugates utilising
natural and unnatural building blocks.3 En route we achieved
interesting conformationally ordered structural architectures
like turns,4 helices,5 sheets,6 folds,7 zipper motifs,8 etc.
Herein, we unveil another striking case of pseudo-β-hairpin
formation by tetrapeptide sequences comprising an Ant–Pro
dipeptide (at the loop region) adjoining sheet promoting
amino acids valine and leucine at the N- and C-termini,
respectively. These tetrapeptides are shown to exhibit two
inter-residual hydrogen-bonding viz. (i → i + 1)-type C9 and
(i − 2 ← i + 3)-type C17 networks, between the central and
terminal residues, respectively, and are anticipated to promote
anti-parallel β-sheet formation.
The Ant–Pro unit is evidently known to exhibit a unique
nine-membered H-bonding interaction between the amide of
Ant and CvO of Pro units, driven by the sterically enforced
orthogonal arrangement of the rings.4a This characteristic
feature of this dipeptide unit is in contrast to the native
β-turns that display a backward hydrogen-bonding pattern
(1 ← 4)-type involving four residues. A juxtaposition of the native
β-hairpin and the pseudo-β-hairpin demonstrating the dispa-
rate H-bonding networks has been shown in Fig. 1 (vide infra).
An exhaustive analysis via structural and chirality perturbation
in and around the turn segment confirmed the strong reverse
turn inducing nature of the Ant–Pro motif.11 Incidentally, the
observed Pro ϕ and φ dihedral angles in the Ant–Pro reverse-
turn motif described herein are typical of the polyproline II
semi-extended conformation.12 Conjoining this dipeptide
segment with the sheet preferring amino acids led to the for-
mation of β-hairpin mimics with two unusual H-bonding
patterns.
Stereochemical alteration is known to cause conformational
modulation in different secondary structures, thus serving as a
good means to assess the stability and conformational propen-
sity of different peptide chains.13 Therefore, we designed, syn-
thesized and evaluated the hairpin nucleation tendencies of
different stereochemically reordered14 tetrapeptide sequences:
R1CO-L/DVal-Ant-L/DPro-L/DLeu-NHR2, comprising the Ant–Pro
turn motif at the loop region (Fig. 2). Derivatives with varying
aDivision of Organic Synthesis, National Chemical Laboratory,
Dr. Homi Bhabha Road, Pune 411 008, India. E-mail: gj.sanjayan@ncl.res.in
http://nclwebapps.ncl.res.in/gjsanjayan; Fax: (+91) 020-25893153;
Tel: (+91) 020-25902082
bCentral Material Characterization Division, National Chemical Laboratory,
Dr. Homi Bhabha Road, Pune 411 008, India
cCentral NMR Facility, National Chemical Laboratory, Dr. Homi Bhabha Road,
Pune 411 008, India
†Electronic supplementary information (ESI) available: Full experimental proce-
dures, 1H NMR, 13C NMR, and ESI mass spectra of all new compounds. CCDC
964584. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c3ob42016g
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stereochemical patterns of α-amino acids viz. LLL (homo-
chiral) and DLL (heterochiral), LDL and DLD (heterochiral
with alternating stereochemistry) were synthesized and their
structural pre-disposition was explored. In order to carry out
the solution phase investigations, pivolyl-protection was pre-
ferred that confers structural rigidity at the N-terminus and
methylamide protection at the C-terminus for a distinct non-
overlapping signal.
Results and discussion
Synthesis
Fig. 1 Schematic representation with intramolecular H-bonding pat-
terns with their directions (highlighted with shaded arrows) of a typical
β-hairpin motif found in proteins (left) and a novel pseudo-β-hairpin fea-
turing the Ant–Pro reverse turn (right). Note that there are distinctive
directional changes in the H-bonding pattern in the native hairpin and
pseudo-β-hairpin reported herein.
Syntheses of all the tetrapeptide analogues 1–4 were carried
out using conventional solution phase peptide synthesis under
standard coupling conditions (Scheme 1). Coupling reactions
were carried out from the N-terminal end in order to avert the
formation of the benzoxazinone intermediate obtained on acti-
vation of the anthranilic carboxylic group.7a,11a,15 After synthe-
sizing dipeptides 8a, 8c and 8e, their corresponding amines
were coupled with Boc-protected anthranilic acid 9 using
HBTU as the coupling agent in the presence of DIEA as a base
to afford tripeptides 10a, 10c and 10e, respectively. Free amine
liberated by deprotection of 10a was coupled with Boc-LVal-OH
and Boc-DVal-OH to afford tetrapeptides 1a and 2a, respect-
ively. The N-terminus of tetrapeptide analogues was converted
into their corresponding pivaloyl analogs 1c and 2c,
which were then subjected to amidation by treating it with
Fig. 2 Designed Ant–Pro oligomers with chirality modulation.
Scheme 1 Reagents and conditions: (i) DCC, DMAP, DCM, 12 h; (ii) TFA–DCM, 1 : 1, 2 h; (iii) HBTU, DIEA, DCM, 12 h; (iv) Piv-Cl, Et3N, DCM, 0 °C, rt,
2 h; (v) MeNH2, MeOH, 4 h; (vi) LiOH·2H2O, THF–H2O, 6 h; (vii) H2N-(p)C6H4-Br, HBTU, DIEA, DCM, 12 h; (viii) TEA, DCM, 0 °C, rt, 1 h.
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methanolic methylamine solution to afford 1 and 2, respect- corresponding long range nOes were notably weak (Fig. 4a–c).
ively. For analogues 3 and 4, the free amines 10d and 10f were Contrary to the expectations from the downfield NH5 proton
subjected to coupling with phthalimide protected valine. The of compound 2 in its 1H NMR spectrum, the 2D spectral
tetramers thus obtained were treated with methanolic methyl- interpretation revealed the partially bound state of NH5 with
amine for simultaneous phthalimide deprotection and methyl- carbonyl of the Piv-group. But another medium dipolar coup-
amide conversion – followed eventually by N-terminus pivaloyl ling observed between NH5 and ValαH (C2H) confirmed its
protection.
folded orientation (Fig. 4d).
Evaluation of the strength of hydrogen-bonds through NMR
studies. Examination of the strength of these H-bonded con-
tacts from MeOD exchange studies faced problems due to
plausible differential solvation of amide NHs, which caused
the protons to shift downfield and resist NH/D exchange (see
ESI, page S40†).18 Thus, variable temperature NMR exper-
iments at 2 mM concentration for all tetrapeptide sequences
were carried out.19 The comparison of chemical shift values of
NH2 (anthranilamide involved in C9-turn formation) and NH5
(methylamide proton that participates in the C17-H-bonding
network) particularly were thoroughly inspected (see ESI,
pages S41–S49†). Strikingly, a considerably lower temperature
coefficient (ΔδHN/ΔT) of ≈−2.5 ppb K−1 was observed for NH2
of tetrapeptides 1 and 2, but a poor value of >−7.5 ppb K−1 for
NH5 indicated solvent exposition of the terminal amide
proton (see ESI, pages S41–S44†). It is noteworthy that ΔδHN/
ΔT lower than −4 ppb K−1 is suggestive of strong intramole-
cular hydrogen bonding.19
Solution-state structural studies
The interactions arising from the folding pattern of peptides
were evaluated using solution-state NMR studies, which were
undertaken for all the tetramer analogues 1–4. A first glimpse
of the 1H-NMR spectra (CDCl3, 298 K) of all the analogues
suggested the presence of
a nine-membered H-bonding
network from the appearance of anthranilamide NH protons
at about 9–9.5 ppm (Fig. 3).16 Similarly, the chemical shift
values observed for NH5 provisionally provided a basis for the
presence of 17-membered H-bonding interaction.17
In the homochiral tetrapeptide 1, the appearance of a rela-
tively upfield and broad NH5 at 7.4 δ, also revealing concen-
tration dependence (see ESI, page S69, Fig. S17†), clearly
suggested the absence of terminal H-bonding interaction. In
the case of all the heterochiral analogues 2–4, the downfield
chemical shifts for NH5 appearing at >7.9 ppm (with consist-
ent chemical shift values at different concentrations, see ESI,
pages S69–S70, Fig. S18–S20†) were indicative of the presence
of terminal H-bonding interactions.
To accrue further evidence to validate these observations,
we undertook an extensive structural analysis using solution-
state NMR studies. 2D NOESY experiments of all tetrapeptide
sequences unambiguously confirmed the presence of a C9-
turn, evident from the prominent nOe cross-peaks observed
between the amide NH2 vs. ProδH protons (C17/17′H) due to
the folding induced by the 9-membered H-bonding formation
(see ESI, pages S71–S74† for nOe details). Confirmation of the
17-membered H-bonding network was obtained from the diag-
nostic long-range dipolar coupling interactions arising from
the spatial proximity between Piv- (C27H) vs. methylamide
NH5 and methyl (C25H) protons (Fig. 4). The heterochiral ana-
logues 3 and 4 displayed intense cross-peaks owing to both
these terminal interactions (Fig. 4e–h). But, in the case of
homochiral analogue 1 and heterochiral tetrapeptide 2, the
Strangely for 3 and 4, comparable values of about −5 ppb K−1
for both NH2 and NH5 were obtained (see ESI, pages S45–
S48†), revealing both amide protons to be in equilibrium
between hydrogen-bonded and non-hydrogen-bonded states.
These observations indicated the fact that alternate hetero-
chiral analogues 3 and 4 exhibit a better stereochemistry, with
respect to compounds
nucleation.
1 and 2, for possible hairpin
A variable temperature study was undertaken for tetra-
peptide 5, which strangely revealed a temperature coefficient
of −5.9 ppb K−1 (see ESI, page S49†), contradicting the obser-
vations of strong terminal intramolecular H-bonding contact
apparent from its crystal structure (vide infra). These obser-
vations presumably suggest the predominance of packing
effects in crystals. However, the collective interpretation of
solution-state NMR studies suggests that though the hetero-
chiral tetrapeptides 2 and 5 reveal the sign of hairpin nuclea-
tion, the orientation of NH5 poorly drives the chains in a
parallel fashion.
Solid-state analysis of tetramer 5. Extensive efforts culmi-
nated in the obtainment of crystals of heterochiral DLL deriva-
tive 5 (Fig. 5).20 Analogue 5 was synthesized by coupling the
acid counterpart of 2a with 4-bromoaniline. Interestingly, the
crystal structure revealed the presence of a strong C9-turn
L
between anthranilamide NH2 and CvO of the Pro residue in
the i → i + 1 forward direction, supporting the earlier obser-
vations. It also revealed the presence of a 17-membered H-
bonded ring between the CvO of tBoc functionality of Val and
the methylamide functionality of the Leu residue in the i − 2 ←
i + 3 reverse direction. Strikingly, the observed H-bond dis-
tances [d(N–H⋯OvC)] = 2.1 Å and H-bonding geometry of the
Fig. 3 Stacked plot of the amide region of tetramer analogues 1–4 for
the comparison of chemical shift values of NH2 and NH5 amide
protons. Note: Molecular structures of 1–4 are given in Fig. 2.
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Fig. 4 Selected nOe extracts from the 2D NOESY data of 1–4 (CDCl3, 400/500 MHz, 298 K): (a), (b), (e) & (f) Piv vs. methylamide –CH3; (c), (g) & (h)
Piv vs. NH5; (d) NH5 vs. ValαH.
CD spectra were recorded in a non-polar solvent like 2,2,2-
triflouroethanol (TFE) and in a polar competing solvent like
acetonitrile for a comparison (Fig. 6). From the patterns
obtained, the maxima/minima at ca. 218 nm were indicative of
the hairpin-like folding pattern that was consistently observed
in alternate heterochiral analogues 3 and 4 featuring an exact
mirror image of the absorption peaks, irrespective of the
solvent variation.
The homochiral tetrapeptide 1 displayed totally distinct CD
signatures on solvent variation with no defined cotton effect,
though a slight conformational ordering was witnessed in
Fig. 5 Molecular structure (left) and crystal structure (right) of 5.
acetonitrile. Analogues 2 and 5 displayed slight changes in the
absorption values, i.e. with a weak maximum at ≈221 nm in
C9- and C17-membered H-bonds are characterized by the
TFE which appears at 218 nm in acetonitrile with enhanced
angles (N–H⋯O) = 165° and 168°, respectively, confirming
equal strength for both the interactions.
intensity, revealing solvent dependence. Intense absorbance
peaks at around 250 nm were consistently observed for all the
tetrapeptide derivatives owing to the backbone aromatic
groups/aromatic electronic transitions.21
IR and CD studies. IR spectra were recorded in CHCl3 at
different concentrations, which displayed a single strong
absorption band due to the N–H stretch at about ≈3310 cm−1
,
supporting the presence of intramolecularly H-bonded amide
protons of analogues 3 and 4 (see ESI, pages S77–S78†). In con-
trast, tetrapeptide 1 exhibited no sharp band NH stretching,
representing the availability of amide protons for intermole-
cular contacts.8 Analogue 2 on the other hand featured strong
absorption bands at about 3311 cm−1 and 3439 cm−1 confirm-
ing a partially bound state of the amide NHs (see ESI, pages
S75–S76†). These observations were corroborated well by CD
studies which revealed almost similar behaviour.
Conclusion
In conclusion, we have successfully demonstrated the essential
requisites for the design of novel pseudo-β-hairpin mimics
based on the Ant–Pro reverse turn motif. Detailed structural
investigations suggest that configurational control has an
immense role to play in orientating the arms of the hairpin
motif in a parallel fashion to support β-sheet formation.
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(1.89 mL, 10.887 mmol, 1.5 equiv.) and HBTU (3.3 g,
8.709 mmol, 1.2 equiv.) were added at 0 °C. After 12 h, the
reaction mixture was diluted with DCM (30 mL) and washed
sequentially with saturated solutions of NaHCO3 (20 mL),
water (20 mL) and KHSO4 (20 mL). The washings were
extracted with DCM (10 mL × 3) and the combined organic
layer was dried over anhydrous Na2SO4 and evaporated under
reduced pressure to obtain the crude residue, which was puri-
fied by column chromatography (eluent: 30% AcOEt–pet.
ether, Rf: 0.3) to afford 10a as a waxy solid (2.3 g, 85%). [α]D25.3
:
−121.10° (c = 1, CHCl3); IR (CHCl3, ν (cm−1): 3364, 3020, 2977,
1
2401, 1725, 1683, 1624, 1521, 1417, 1159, 1046, 929; H NMR
(CDCl3/200 MHz): δ ppm 8.35 (s, 1H, amide), 8.17–8.13 (d, J =
8.34 Hz, 1H), 7.41–7.31 (m, 2H), 7.04–6.92 (m, 2H), 4.81–4.74
(m, 1H), 4.66–4.54 (m, 1H), 3.73 (s, 3H), 3.57–3.47 (m, 2H),
2.33–1.78 (m, 4H), 1.71–1.60 (m, 2H), 1.50 (s, 9H), 0.93–0.88
(m, 6H); 13C NMR (CDCl3, 50 MHz): δ ppm 173.2, 170.9, 168.9,
153.0, 137.2, 131.0, 127.4, 123.6, 121.8, 120.4, 80.4, 59.6, 52.2,
50.9, 50.6, 41.2, 28.3, 27.7, 25.3, 24.8, 22.7, 21.9; MALDI-TOF/
TOF: 484.8722 (M + Na)+; 500.8892 (M + K)+; elemental analysis
calculated for C24H35N3O6: C, 62.45; H, 7.64; N, 9.10; found:
C, 62.36; H, 7.56; N, 9.18.
(S)-Methyl 2-((R)-1-(2-((tert-butoxycarbonyl)amino)benzoyl)-
pyrrolidine-2-carboxamido)-4-methylpentanoate 10c. Compound
10c was synthesized following the procedure for 10a. The
crude product was purified by column chromatography
(eluent: 30% AcOEt–pet. ether, Rf: 0.3) to furnish 10c (82%) as
a waxy solid. [α]2D5.9: 111.728° (c = 1, CHCl3); IR (CHCl3,
ν (cm−1): 3355, 3017, 2975, 2897, 2401, 1724, 1683, 1625, 1590,
Fig. 6 Comparison of CD spectra of tetramer analogues 1–5 in TFE
(top) and 1–5 in acetonitrile (bottom), respectively (0.1 mM).
Amongst the tetrapeptide sequences evaluated, heterochiral
sequences with alternating stereochemistry exhibited the best
hairpin nucleation propensity. We anticipate that this under-
standing will be deeply beneficial in developing the locale of
β-hairpin mimetics.
1
1521, 1413, 1158, 1050, 928; H NMR (CDCl3/200 MHz): δ ppm
8.23 (s, 1H, amide), 8.11–8.07 (d, J = 8.34 Hz, 1H), 7.42–7.27
(m, 3H), 7.08–7.01 (m, 1H), 4.90–4.84 (m, 1H), 4.67–4.58
(m, 1H), 3.70 (s, 3H), 3.60–3.48 (m, 2H), 2.46–2.38 (m, 1H),
2.15–1.82 (m, 4H), 1.70–1.60 (m, 2H), 1.50 (s, 9H), 0.95–0.93
(m, 6H); 13C NMR (CDCl3, 50 MHz): δ ppm 173.3, 170.9, 170.2,
153.1, 136.9, 131.0, 127.5, 124.3, 122.2, 121.1, 80.4, 59.2, 52.3,
50.9, 50.3, 49.0, 41.2, 28.3, 27.0, 25.2, 24.9, 22.8, 21.7; MALDI-
TOF/TOF: 485.0148 (M + Na)+; 501.0496 (M + K)+; elemental
analysis calculated for C24H35N3O6: C, 62.45; H, 7.64; N, 9.10;
found: C, 62.36; H, 7.56; N, 9.18.
Experimental procedures
Single crystal X-ray crystallographic studies of 5
Single crystals of 5 were grown by slow evaporation of the solu-
tion mixture of ethyl acetate and pet. ether. A colorless needle-
type crystal of approximate size 0.26 × 0.21 × 0.03 mm3 was
used for data collection. Total runs = 3, total frames = 1265,
θ range = 2.13 to 25.00°, completeness to θ of 25.00° is 99.9%,
C34H46BrN5O5, M = 702.69. Crystal system: orthorhombic,
space group P212121, a = 9.6704(5), b = 9.8238(6), c = 38.263(2) Å,
V = 3634.9(3) Å3, Z = 4, Dc = 1.280 g cc−1, µ(Mo-Kα) =
1.179 mm−1, 46 085 reflections measured, 6400 unique reflec-
tions, 5166 observed [I > 2σ(I)], R value 0.0447, wR2 = 0.1143,
(S)-Methyl 2-((R)-1-(2-((tert-butoxycarbonyl)amino)benzoyl)-
pyrrolidine-2-carboxamido)-4-methylpentanoate 10e. Compound
10e was synthesized following the procedure for 10a. The
crude product was purified by column chromatography
(eluent: 30% AcOEt–pet. ether, Rf: 0.3) to furnish 10e (92%) as
a waxy solid. [α]2D6.1: −112.868° (c = 1, CHCl3); IR (CHCl3,
ν (cm−1): 3348, 3018, 2897, 2401, 1724, 1682, 1625, 1590, 1520,
1
1412, 1369, 1158, 1052, 928; H NMR (CDCl3/200 MHz): δ ppm
largest diff. peak and hole 0.45 and −0.21 e Å−3
.
8.23 (s, 1H, amide), 8.10–8.06 (d, J = 8.34 Hz, 1H), 7.41–7.27
(m, 3H), 7.07–7.00 (m, 1H), 4.89–4.80 (m, 1H), 4.66–4.55
(m, 1H), 3.69 (s, 3H), 3.53–3.44 (m, 2H), 2.45–2.32 (m, 1H),
2.15–1.82 (m, 4H), 1.69–1.55 (m, 2H), 1.49 (s, 9H), 0.94–0.91
General method for the synthesis of compounds 10a, 10c
and 10e
(S)-Methyl 2-((S)-1-(2-((tert-butoxycarbonyl)amino)benzoyl)- (m, 6H); 13C NMR (CDCl3, 50 MHz): δ ppm 173.3, 170.9, 170.1,
pyrrolidine-2-carboxamido)-4-methylpentanoate 10a. To a solu- 153.1, 136.9, 131.0, 127.5, 124.2, 122.2, 121.0, 80.4, 59.2, 52.3,
tion of compound 8b (1.8 g, 7.258 mmol) and Boc-Ant-OH 50.8, 50.3, 41.2, 28.3, 27.0, 25.2, 24.8, 22.8, 21.7; MALDI-TOF/
(1.37 g, 5.806 mmol, 0.8 equiv.) in DCM (30 mL), DIPEA TOF: 484.8722 (M + Na)+; 500.8892 (M + K)+; elemental analysis
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calculated for C24H35N3O6: C, 62.45; H, 7.64; N, 9.10; found: pentanoic acid 2e. To a solution of 2a (0.5 g, 0.091 mmol,
C, 62.43; H, 7.69; N, 8.98. 1 equiv.) in THF–H2O (2 : 1 v/v), LiOH·2H2O (0.01 g, 0.182 mmol,
(S)-Methyl 2-((S)-1-(2-((S)-2-((tert-butoxycarbonyl)amino)- 2 equiv.) was added and the reaction mixture was stirred
3-methylbutanamido)benzoyl)pyrrolidine-2-carboxamido)-4- for 12 h. After completion of the reaction, THF was removed
methylpentanoate 1a. To a solution of compound 10b (0.4 g, in vacuo and the solution was neutralized using KHSO4 solu-
1.108 mmol, 1 equiv.) and Boc-LVal-OH (0.29 g, 1.329 mmol, tion. The crude product was extracted into ethyl acetate and
1.2 equiv.) in DCM (5 mL), HBTU (0.546 g, 1.44 mmol, was washed with brine solution. The organic layer was dried
1.3 equiv.) and DIEA (0.287 mL, 1.662 mmol, 1.5 equiv.) were over Na2SO4 and evaporated in vacuo to afford 2e. The crude
added. After 12 h, the reaction mixture was diluted with DCM product obtained was used for the next step without further
(30 mL) and washed sequentially with saturated solutions of purification.
NaHCO3 (20 mL), water (20 mL) and KHSO4 (20 mL). The
washings were extracted with DCM (10 mL × 3) and the com- 4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carbonyl)-
bined organic layer was dried over anhydrous Na2SO4 and phenyl)amino)-3-methyl-1-oxobutan-2-yl)carbamate 5. To
tert-Butyl ((R)-1-((2-((S)-2-(((S)-1-((4-bromophenyl)amino)-
a
evaporated under reduced pressure to obtain the crude solution of acid 2e (0.1 g, 0.183 mmol, 1 equiv.) and 4-bromo-
residue, which was purified by column chromatography aniline (0.031 g, 0.183 mmol, 1 equiv.) in dry DCM (10 mL),
(eluent: 40% AcOEt–pet. ether, Rf: 0.4) to afford 1a (0.40 g, HBTU (0.083 g, 0.219 mmol, 1.2 equiv.) and DIEA (0.041 mL,
65%) as a sticky liquid. [α]2D6.9: −44.216° (c = 1, CHCl3); IR 0.238 mmol, 1.3 equiv.) were added and the reaction was main-
(CHCl3, ν (cm−1): 3280, 3069, 2963, 2931, 2875, 1745, 1715, tained at 0 °C. The mixture was stirred at room temperature
1668, 1589, 1541, 1457, 1421, 1391, 1370, 1275, 1247, 1162, for an additional 12 h. DCM (10 mL) was added to the reaction
1092, 1016, 987, 873; 1H NMR (CDCl3/200 MHz): δ ppm 9.39 mixture and the combined organic layers were washed sequen-
(s, 1H), 8.26–8.28 (d, J = 7.96 Hz, 1H), 7.37–7.21 (m, 2H), tially with solutions of KHSO4, NaHCO3 and brine. Organic
7.09–7.02 (m, 1H), 6.78–6.74 (d, J = 7.96 Hz, 1H), 5.34–5.30 layer was then dried over Na2SO4 and was evaporated in vacuo.
(d, J = 8.97 Hz, 1H), 4.68–4.53 (m, 2H), 4.28–4.21 (m, 1H), 3.68 The crude product was purified by column chromatography
(s, 3H), 3.48–3.17 (m, 2H), 2.21–2.11 (m, 3H), 1.98–1.5 (m, 5H), (eluent: 40% AcOEt–pet. ether, Rf: 0.4) to furnish 5 (0.75 g,
1.36 (s, 9H), 0.97–0.94 (d, J = 6.69 Hz, 3H), 0.88–0.84 (m, 9H); 60%) as a white crystalline solid. Mp: 229–231 °C; [α]D27.1
:
13C NMR (CDCl3, 50 MHz): δ ppm 173.6, 171.6, 168.5, 165.6, −73.588° (c 1, CHCl3); IR (CHCl3) ν (cm−1): 3272, 2962, 2930,
155.9, 134.8, 130.4, 126.6, 124.0, 121.9, 79.4, 68.6, 60.2, 59.9, 2403, 1682.4, 1621, 1538, 1490, 1457, 1394, 1302, 1246, 1163,
52.3, 51.0, 49.1, 43.9, 41.2, 31.3, 29.2, 28.2, 25.1, 24.7, 1075, 1010, 828; 1H NMR (CDCl3, 500 MHz): δ ppm 9.64 (s, 1H,
22.7, 21.8, 19.3, 17.5; MALDI-TOF/TOF: 584.1642 (M + Na)+; amide), 9.58 (s, 1H, amide), 8.34–8.32 (d, J = 8.24 Hz, 1H),
600.1794 (M
C29H44N4O7: C, 62.12; H, 7.91; N, 9.99; found: C, 62.15; 7.14–7.09 (m, 2H), 5.56–5.54 (d, J = 9.46 Hz, 1H), 4.96–4.88
H, 7.69; N, 9.88. (dd, J = 6.71 Hz, J = 9.16 Hz, 1H), 4.72–4.69 (m, 1H), 4.60–4.56
+
K)+; elemental analysis calculated for 7.59–7.57 (m, 2H), 7.39–7.37 (m, 3H), 7.24–7.22 (m, 3H),
(S)-Methyl 2-((S)-1-(2-((R)-2-((tert-butoxycarbonyl)amino)- (m, 1H), 3.39–3.31 (m, 2H), 2.30–2.24 (m, 1H), 2.07–1.84 (m,
3-methylbutanamido)benzoyl)pyrrolidine-2-carboxamido)-4- 4H), 1.74–1.64 (m, 3H), 1.46 (s, 9H), 0.98–0.96 (d, J = 6.10 Hz,
methylpentanoate 2a. Compound 2a was synthesized follow- 3H), 0.91–0.86 (m, 9H); 13C NMR (CDCl3, 125 MHz): δ ppm
ing the procedure for 1a. The crude product was purified by 173.1, 171.6, 171.0, 168.6, 156.1, 137.6, 134.5, 131.6, 130.0,
column chromatography (eluent: 40% AcOEt–pet. ether, Rf: 127.0, 125.3, 123.9, 121.3, 121.25, 116.5, 79.9, 59.8, 58.9, 54.0,
0.4) to furnish 2a (75%) as a sticky liquid. [α]2D6.9: −66.352° (c = 49.2, 40.9, 33.3, 29.6, 28.5, 24.8, 22.6, 22.3, 18.9, 18.4; MALDI-
1, CHCl3); IR (CHCl3, ν (cm−1): 3300, 2960, 2925, 1747, 1667, TOF/TOF: 725.4464 (M + Na)+; 741.5165 (M + K)+; Anal. calcd
1626, 1514, 1456, 1414, 1368, 1246, 1204, 1160, 1017, 875; IR for C34H46BrN5O6: C, 58.28; H, 6.62; Br, 11.40; N, 10.00; found:
(CHCl3, ν (cm−1): 3280, 3069, 2963, 2931, 2875, 1745, 1715, C, 58.36; H, 6.69; Br, 11.41; N, 9.94.
1668, 1589, 1541, 1457, 1421, 1391, 1370, 1275, 1247, 1162,
General method for the synthesis of compounds 3a and 4a
1092, 1016, 987, 873; 1H NMR (CDCl3/200 MHz): δ ppm 9.22
(s, 1H), 8.39–8.35 (d, J = 8.21 Hz, 1H), 7.41–7.26 (m, 2H),
(S)-Methyl 2-((R)-1-(2-((S)-2-(1,3-dioxoisoindolin-2-yl)-
7.11–7.03 (m, 1H), 6.97–6.93 (bs, 1H), 5.82–5.77 (d, J = 8.84 Hz, 3-methylbutanamido)benzoyl)pyrrolidine-2-carboxamido)-
1H), 4.71–4.54 (m, 2H), 4.19–4.12 (m, 1H), 3.72 (s, 3H), 4-methylpentanoate 3a. To a solution of Phth-LVal-OH (0.576 g,
3.42–3.37 (m, 2H), 2.26–2.19 (m, 4H), 2.02–1.51 (m, 4H), 1.40 2.333 mmol, 1.2 equiv.) in dry DCM (10 mL) and cat. amount
(s, 9H), 0.99–0.87 (m, 12H); 13C NMR (CDCl3, 50 MHz): δ ppm of DMF, oxalyl chloride (0.20 mL, 2.527 mmol, 1.3 equiv.) was
173.1, 171.5, 171.0, 168.6, 165.6, 155.6, 135.3, 130.6, 126.8, added dropwise at 0 °C. Later, the reaction was allowed to con-
125.3, 123.4, 121.3, 79.3, 61.2, 59.7, 52.1, 51.1, 49.6, 41.0, 31.1, tinue at rt for 1 h. DCM was then stripped off in vacuo. The
28.7, 28.2, 25.2, 24.6, 22.6, 21.7, 19.2, 17.8; MALDI-TOF/TOF: solution containing 10d (0.7 g, 1.944 mmol, 1 equiv.) and Et3N
584.0563 (M + Na)+; 600.0703 (M + K)+; elemental analysis (0.394 mL, 2.916 mmol, 1.5 equiv.) in dry DCM (15 mL) was
calculated for C29H44N4O7: C, 62.12; H, 7.91; N, 9.99; found: cooled in an ice bath with stirring. A solution of Phth-LVal-
C, 62.20; H, 7.72; N, 9.91.
COCl in DCM (10 mL) was added dropwise for 15 min to the
(S)-2-((S)-1-(2-((R)-2-((tert-Butoxycarbonyl)amino)-3-methyl above reaction mixture. The mixture was stirred at room temp-
butanamido)benzoyl)pyrrolidine-2-carboxamido)-4-methyl erature for an additional 1 h. DCM (10 mL) was added to the
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mixture which was then washed with KHSO4 solution (10 mL), [α]2D5.7: −53.748° (c 1, CHCl3); IR (CHCl3) ν (cm−1): 3408, 3020,
saturated aqueous NaHCO3 (10 mL), and brine (10 mL). The 2400, 1635, 1420, 1318, 1079; 1H NMR (CDCl3/200 MHz):
organic layer was dried over Na2SO4 and evaporated in vacuo. δ ppm 9.44 (s, 1H), 8.25–8.21 (d, J = 7.96 Hz, 1H), 7.42–7.26
The crude product was purified by column chromatography (m, 2H), 7.16–7.11 (m, 1H), 7.0–6.96 (d, J = 8.46 Hz, 1H),
(eluent: 40% AcOEt–pet. ether, Rf: 0.4) to furnish 3a (0.96 g, 6.43–6.39 (d, J = 8.59 Hz, 1H), 4.76–4.55 (m, 3H), 3.73 (s, 3H),
85%) as a white crystalline solid. Mp: 157–160 °C; [α]D26.9
:
3.46–3.21 (m, 2H), 2.28–2.12 (m, 2H), 2.03–1.51 (m, 6H), 1.22
−117.856° (c 1, CHCl3); IR (CHCl3) ν (cm−1): IR (CHCl3) (s, 9H), 1.02–0.98 (d, J = 6.69 Hz, 3H), 0.93–0.85 (m, 9H);
ν (cm−1): 3063, 2959, 2927, 2874, 1722, 1622, 1532, 1453, 1417, 13C NMR (CDCl3, 50 MHz): δ ppm 178.6, 173.8, 171.7, 171.3,
1384, 1247, 1153, 1071, 988; 1H NMR (200 MHz, CDCl3) δ: 9.54 168.4, 134.6, 130.4, 127.0, 126.7, 124.1, 122.1, 60.5, 58.3, 52.3,
(1H, amide), 8.08–8.04 (d, J = 8.08 Hz, 1H), 7.91–7.85 (m, 2H), 50.8, 49.1, 41.2, 38.8, 31.2, 29.6, 29.2, 27.4, 25.1, 24.9, 22.7,
7.78–7.74 (m, 2H), 7.43–7.33 (m, 2H), 7.17–7.10 (m, 2H), 21.7, 19.3, 17.8; MALDI-TOF/TOF: 570.2427 (M + K)+; Anal.
4.63–4.52 (m, 3H), 3.69 (s, 3H), 3.50–3.42 (m, 2H), 3.04–2.92 calcd for C29H44N4O6: C, 63.95; H, 8.14; N, 10.29; found:
(m, 1H), 2.46–2.35 (m, 1H), 2.1–1.92 (m, 2H), 1.85–1.46 (m, C, 63.88; H, 8.20; N, 10.25.
4H), 1.19–1.16 (d, J = 6.57 Hz, 3H), 0.93–0.90 (m, 9H); 13C NMR
(S)-Methyl 4-methyl-2-((S)-1-(2-((R)-3-methyl-2-pivalamido
(50 MHz, CDCl3) δ: 173.1, 170.6, 170.2, 168.0, 166.8, 135.2, butanamido)benzoyl)pyrrolidine-2-carboxamido)pentanoate 2c.
134.3, 131.6, 130.9, 127.5, 126.2, 124.0, 123.5, 123.2, 61.6, 59.0, Compound 2c was synthesized following the procedure for 1c
52.2, 50.8, 50.4, 41.0, 38.6, 29.6, 27.5, 26.8, 25.2, 24.8, 22.8, from 2b. The crude product was purified by column chromato-
21.8, 20.5, 19.4; MALDI-TOF: 613.3940 (M + Na)+, 629.4184 graphy (eluent: 45% AcOEt–pet. ether, Rf: 0.4) to furnish 2c
(M + K)+; Anal. calcd for C32H38N4O7: C, 64.85; H, 6.80; N, 9.45; (86%) as a sticky liquid. Mp: 58–60 °C; [α]2D6.3: −89.084° (c 1,
found: C, 64.98; H, 6.69; N, 9.49.
CHCl3); IR (CHCl3) ν (cm−1): 3283, 3073, 2960, 2873, 1747,
(R)-Methyl 2-((S)-1-(2-((R)-2-(1,3-dioxoisoindolin-2-yl)-3-methyl- 1667, 1626, 1588, 1524, 1455, 1417, 1370, 1301, 1201, 1155,
1
butanamido)benzoyl)pyrrolidine-2-carboxamido)-4-methyl- 1025, 986, 914; H NMR (CDCl3/200 MHz): δ ppm 9.53 (s, 1H),
pentanoate 4a. Compound 4a was synthesized following the 8.32–8.28 (d, J = 8.21 Hz, 1H), 7.44–7.32 (m, 2H), 7.15–7.07 (m,
procedure for 3a. The crude product was purified by column 1H), 6.82–6.79 (d, J = 7.83 Hz, 1H), 6.51–6.47 (d, J = 8.21 Hz,
chromatography (eluent: 40% AcOEt–pet. ether, Rf: 0.4) to 1H), 4.80–4.73 (m, 1H), 4.63–4.49 (m, 2H), 3.74 (s, 3H),
furnish 4a (86%) as a white solid. Mp: 151–153 °C; [α]D26.2
:
3.58–3.42 (m, 2H), 2.31–2.16 (m, 3H), 2.04–1.6 (m, 5H), 1.23
−107.592° (c 1, CHCl3); IR (CHCl3) ν (cm−1): 3062, 2959, 2923, (s, 9H), 1.02–0.89 (m, 12H); 13C NMR (CDCl3, 50 MHz):
2873, 2951, 1721, 1623, 1532, 1456, 1428, 1383, 1249, 1153, ppm 178.4, 173.3, 171.1, 170.4, 169.2, 135.9, 131.0,
δ
1071; 1H NMR (200 MHz, CDCl3) δ: 9.55 (1H, amide), 8.08–8.04 127.3, 124.8, 123.4, 121.9, 59.7, 58.6, 52.3, 51.1, 50.5, 41.1,
(d, J = 7.96 Hz, 1H), 7.91–7.87 (m, 2H), 7.79–7.75 (m, 2H), 38.9, 31.8, 29.6, 28.1, 27.5, 25.3, 24.8, 22.7, 21.9, 19.3,
7.44–7.34 (m, 2H), 7.17–7.10 (m, 2H), 4.63–4.53 (m, 3H), 3.69 17.7; MALDI-TOF/TOF: 570.1554 (M + K)+; Anal. calcd for
(s, 3H), 3.51–3.42 (m, 2H), 3.04–2.93 (m, 1H), 2.49–2.32 C29H44N4O6: C, 63.95; H, 8.14; N, 10.29; found: C, 63.88;
(m, 1H), 2.07–1.93 (m, 2H), 1.86–1.47 (m, 4H), 1.19–1.16 (d, H, 8.20; N, 10.25.
J = 6.69 Hz, 3H), 0.94–0.90 (m, 9H); 13C NMR (50 MHz, CDCl3)
(S)-N-((S)-4-Methyl-1-(methylamino)-1-oxopentan-2-yl)-1-
δ: 173.1, 170.6, 170.2, 168.0, 166.9, 135.2, 134.32, 131.6, 130.9, (2-((S)-3-methyl-2-pivalamidobutanamido)benzoyl)pyrrolidine-
127.5, 126.3, 124.0, 123.5, 123.3, 61.6, 59.1, 52.2, 50.9, 50.4, 2-carboxamide 1. Compound 1c (0.2 g, 0.25 mmol) was stirred
41.0, 38.6, 27.6, 26.8, 25.2, 24.8, 22.8, 21.8, 20.5, 19.5; MALDI- in saturated solution of methanolic methylamine for 2 h at rt.
TOF: 613.7018 (M + Na)+, 629.7104 (M + K)+; Anal. calcd for Solvent was evaporated in vacuo and the crude product was
C32H38N4O7: C, 64.85; H, 6.80; N, 9.45; found: C, 64.97; purified by column chromatography (eluent: 45% AcOEt–pet.
H, 6.71; N, 9.51.
ether, Rf: 0.4) to furnish 1 (95%) as a white fluffy solid.
Mp: 100–102 °C; [α]D27.1: −50.672° (c 0.5, CHCl3); IR (CHCl3)
ν (cm−1): 3440, 3318, 3020, 2967, 2401, 1648, 1589, 1509, 1457,
General method for the synthesis of compounds 1c and 2c
(S)-Methyl 4-methyl-2-((S)-1-(2-((S)-3-methyl-2-pivalamido 1420, 1369; 1H NMR (400 MHz, CDCl3) δ: 9.68 (1H, amide),
butanamido)benzoyl)pyrrolidine-2-carboxamido)pentanoate 1c. 8.32–8.29 (d, J = 8.28 Hz, 1H), 7.42–7.35 (m, 3H), 7.28–7.26 (m,
To a solution of 1b (0.33 g, 0.713 mmol, 1 equiv.) in dry DCM 1H), 7.16–7.12 (t, J = 7.53 Hz, 1H), 6.50–6.48 (d, J = 8.28 Hz,
(5 mL), Et3N (0.145 mL, 1.071 mmol, 1.5 equiv.) was added. 1H), 4.98–4.94 (m, 1H), 4.74–4.71 (dd, J = 5.27 Hz, J = 8.03 Hz,
The reaction mixture was kept under a N2 atmosphere and 1H), 4.64–4.58 (m, 1H), 3.37–3.23 (m, 2H), 2.78–2.77 (d, J =
the temperature was maintained at 0 °C. Piv-Cl (0.1 mL, 4.52 Hz, 3H), 2.35–2.26 (m, 1H), 2.17–1.07 (m, 3H), 1.85–1.80
0.855 mmol, 1.2 equiv.) was added dropwise slowly into the (m, 1H), 1.70–1.57 (m, 3H), 1.23 (s, 9H), 1.02–1.0 (d, J =
reaction mixture. After 15 min, reaction was allowed to con- 6.78 Hz, 3H), 0.96–0.91 (m, 9H); 13C NMR (100 MHz, CDCl3)
tinue at rt for 1 h. DCM (10 mL) was added to the reaction δ: 178.4, 173.9, 171.8, 171.3, 168.6, 134.5, 130.1, 127.7, 125.9,
mixture and the DCM layer was washed with NaHCO3 solution 124.1, 121.6, 60.5, 58.0, 52.2, 48.9, 40.4, 38.8, 32.3, 29.8, 29.7,
followed by water and brine. The DCM layer was dried over 27.5, 26.4, 24.7, 24.6, 22.7, 22.3, 19.3, 18.2; MALDI-TOF:
Na2SO4 and was concentrated in vacuo. The crude product was 567.2104 (M + Na)+, 583.2536 (M + K)+; Anal. calcd for
purified by column chromatography (eluent: 45% AcOEt–pet. C29H45N5O5: C, 64.06; H, 8.34; N, 12.88; found: C, 64.17;
ether, Rf: 0.4) to furnish 1c (0.30 g, 80%) as a sticky liquid. H, 8.44; N, 12.87.
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(S)-N-((S)-4-Methyl-1-(methylamino)-1-oxopentan-2-yl)-1- J = 8.54 Hz, 1H), 7.24–7.22 (m, 2H), 7.13–7.10 (m, 1H),
(2-((R)-3-methyl-2-pivalamidobutanamido)benzoyl)pyrrolidine- 6.53–6.51 (d, J = 9.16 Hz, 1H), 5.10–5.06 (dd, J = 6.71 Hz, J =
2-carboxamide 2. Compound 2 was synthesized following the 9.16 Hz, 1H), 4.94–4.89 (m, 1H), 4.72–4.69 (m, 1H), 3.40–3.31
procedure for 1 from 2c. The crude product was purified by (m, 2H), 2.77–2.76 (d, J = 4.88 Hz, 3H), 2.37–2.30 (m, 1H),
column chromatography (eluent: 45% AcOEt–pet. ether, Rf: 2.11–1.88 (m, 4H), 1.7–1.61 (m, 3H), 1.22 (s, 9H), 1.03–1.02 (d,
0.4) to furnish 2 (67%) as a sticky liquid. [α]D27.1: −79.944° J = 6.71 Hz, 3H), 0.98–0.95 (m, 9H); 13C NMR (125 MHz,
(c 0.5, CHCl3); IR (CHCl3) ν (cm−1): 3439, 3314, 3020, 2965, CDCl3) δ: 178.2, 172.6, 172.2, 171.1, 168.4, 134.6, 129.8, 127.5,
2874, 2401, 1646, 1589, 1550, 1515, 1456, 1417, 1370, 1296; 125.2, 123.8, 121.6, 60.1, 57.4, 51.8, 49.1, 42.0, 38.9, 33.1, 30.1,
1H NMR (500 MHz, CDCl3) δ: 9.89 (1H, amide), 8.59 (m, 1H, 27.6, 26.3, 25.0, 24.6, 22.7, 22.3, 19.2, 18.0; MALDI-TOF:
amide), 8.40–8.38 (d, J = 8.54 Hz, 1H), 7.44–7.37 (m 2H), 566.8729 (M + Na)+, 583.0687 (M + K)+; Anal. calcd for
7.24–7.23 (d, J = 7.32 Hz, 1H), 7.14–7.11 (m, 1H), 6.60–6.58 (d, C29H45N5O5: C, 64.06; H, 8.34; N, 12.88; found: C, 64.09;
J = 8.54 Hz, 1H), 5.27–5.24 (dd, J = 6.71 Hz, J = 8.54 Hz, 1H), H, 8.39; N, 12.77.
4.78–4.77 (m, 1H), 4.48–4.47 (m, 1H), 3.37–3.32 (m, 2H),
2.8–2.8 (d, J = 2.75 Hz, 3H), 2.33 (m, 1H), 2.08–1.96 (m, 3H),
1.98–1.88 (m, 1H), 1.79–1.63 (m, 3H), 1.22 (s, 9H), 0.99–0.96
Acknowledgements
(m, 9H), 0.93–0.92 (d, J = 6.1 Hz, 3H); 13C NMR (125 MHz,
This paper is dedicated to Professor Goverdhan Mehta on the
CDCl3) δ: 178.3, 174.0, 172.9, 171.0, 168.4, 134.5, 129.9, 127.2,
occasion of his 70th birthday. This work was funded by NCL-I-
125.4, 123.9, 121.0, 59.7, 57.3, 53.3, 49.2, 40.6, 38.9, 33.4, 28.8,
GIB-JRI. RV, ASK and RLG thank CSIR, New Delhi, for research
27.6, 26.4, 24.84, 24.82, 22.7, 22.2, 18.9, 18.3; MALDI-TOF:
fellowships.
566.9368 (M + Na)+, 583.1334 (M + K)+; Anal. calcd for
C29H45N5O5: C, 64.06; H, 8.34; N, 12.88; found: C, 64.09;
H, 8.39; N, 12.77.
(R)-N-((S)-4-Methyl-1-(methylamino)-1-oxopentan-2-yl)-1-
Notes and references
(2-((S)-3-methyl-2-pivalamidobutanamido)benzoyl)pyrrolidine-
2-carboxamide 3. Compound 3 was synthesized following the
procedure for 1 to afford the free amine which was further pro-
tected with the pivaloyl group following the procedure for 1c.
The crude product was purified by column chromatography
(eluent: 45% AcOEt–pet. ether, Rf: 0.4) to furnish 3 (79%) as a
white solid. Mp: 80–82 °C; [α]2D6.1: 22.608° (c 0.5, CHCl3); IR
(CHCl3) ν (cm−1): 3312, 3019, 2966, 2874, 2401, 1648, 1588,
1518, 1456, 1420, 1369, 1297; 1H NMR (500 MHz, CDCl3) δ: 9.7
(1H, amide), 8.32–8.30 (dd, J = 1.53 Hz, J = 8.54 Hz, 1H),
7.91–7.90 (m, 1H, amide), 7.39–7.36 (dd, J = 1.53 Hz, J =
8.54 Hz, 1H), 7.24–7.22 (dd, J = 1.53 Hz, J = 7.32 Hz, 1H),
7.15–7.10 (m, 2H), 6.53–6.51 (d, J = 9.16 Hz, 1H), 5.09–5.06
(dd, J = 7.02 Hz, J = 8.85 Hz, 1H), 4.94–4.90 (m, 1H), 4.72–4.69
(m, 1H), 3.40–3.31 (m, 2H), 2.77–2.76 (d, J = 4.58 Hz, 3H),
2.36–2.29 (m, 1H), 2.12–1.86 (m, 4H), 1.7–1.61 (m, 3H), 1.23 (s,
9H), 1.03–1.02 (d, J = 6.71 Hz, 3H), 0.98–0.95 (m, 9H); 13C NMR
(125 MHz, CDCl3) δ: 178.2, 172.5, 172.2, 171.1, 168.4, 134.7,
129.8, 127.5, 125.2, 123.8, 121.7, 60.1, 57.4, 51.9, 49.2, 42.0,
38.9, 33.2, 30.1, 29.7, 27.7, 26.3, 25.0, 24.6, 22.8, 22.4, 19.3,
18.0; MALDI-TOF: 566.7384 (M + Na)+, 583.7939 (M + K)+; Anal.
calcd for C29H45N5O5: C, 64.06; H, 8.34; N, 12.88; found:
C, 64.09; H, 8.39; N, 12.77.
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2-carboxamide 4. Compound 4 was synthesized following the
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:
(c 0.5, CHCl3); IR (CHCl3) ν (cm−1): 3312, 3018, 2966, 2874,
1
2401, 1648, 1588, 1551, 1513, 1456, 1420, 1369, 1297; H NMR
(500 MHz, CDCl3) δ: 9.71 (1H, amide), 8.32–8.31 (d, J = 8.24 Hz,
1H), 7.93–7.92 (m, 1H, amide), 7.39–7.36 (dd, J = 1.53 Hz,
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782 | Org. Biomol. Chem., 2014, 12, 774–782
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