Synthesis of Psychrophilin e

Article abstract of DOI:10.1021/acs.joc.6b01369

The first total synthesis of psychrophilin E, a potent antiproliferative cyclic tripeptide isolated from Aspergillus versicolor ZLN-60, is reported herein. Key features of the synthesis include the installation of an amide bond between the indole-nitrogen of tryptophan and an anthranilic acid residue, and a high yielding macrolactamization of the linear tripeptide to the desired macrocycle.

Full text of DOI:10.1021/acs.joc.6b01369

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Article
Synthesis of Psychrophilin E
Sarah T. Y. Ngen, Harveen Kaur, Paul Alexander Hume, Daniel P. Furkert, and Margaret A. Brimble
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01369 • Publication Date (Web): 21 Jul 2016
Downloaded from http://pubs.acs.org on July 22, 2016
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Synthesis of Psychrophilin E
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Sarah T. Y. Ngen, Harveen Kaur, Paul A. Hume, Daniel P. Furkert and Margaret A.
Brimble*
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School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland,
New Zealand
Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, 3
Symonds Street, Auckland, New Zealand
m.brimble@auckland.ac.nz
ABSTRACT
The first total synthesis of psychrophilin E, a potent anti-proliferative cyclic tripeptide
isolated from Aspergillus versicolor ZLN-60, is reported herein. Key features of the synthesis
include the installation of an amide bond between the indole-nitrogen of tryptophan and an
anthranilic acid residue, and a high yielding macrolactamization of the linear tripeptide to the
desired macrocycle.
Introduction
In 2004, Dalsgaard et al. discovered a novel family of structurally related nitrated cyclic
peptides, psychrophilins A-D (1-4), which were isolated from the psychrotolerant fungi
Penicillium ribeum and Penicillium algidum (Figure 1).1-3 In 2014, four structurally similar
cyclic des-nitropeptides, psychrophilins E-H (5-8), were further discovered by Peng et al.4
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from the marine-derived fungus Aspergillus versicolor ZLN-60. Psychrophilin D (4) exhibits
moderate cytotoxic activity against P388 murine leukaemia cells (ID50 = 10.1 µg/mL),3 whilst
psychrophilin G (7) exhibits potent lipid-lowering effects in HepG2 hepatocarcinomal cells
(IC50 = 10 µg/mL).4 Interestingly, the isolation of psychrophilin E (5) was simultaneously
reported by Ebada et al.5 from the same fungal strain of the genus Aspergillus, and was
shown to demonstrate potent anti-proliferative activity towards the HCT116 (colon) cell line
(IC50 = 28.5 µg/mL) with high selectivity. Importantly, psychrophilin E (5) demonstrated
more potent cytotoxic activity than cisplatin, a well-known chemotherapeutic agent used
clinically (IC50 = 33.4 µg/mL).5 In contrast, psychrophilins A-C, F and H (1-3, 6, 8) did not
display any biological activity.
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Figure 1. Structures of psychrophilins A-H (1-8).
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Psychrophilins A-H (1-8) are 13-membered macrocycles that possess an unusual amide
linkage between the indole-nitrogen of tryptophan and the anthranilic acid residues.
Interestingly, psychrophilins A-D (1-4) contain an unprecedented nitro-tryptophan moiety
and are the only naturally occurring cyclic peptides known to possess a nitro group.
Furthermore, to the extent of our knowledge, the synthesis of an amide bond between the
indole-nitrogen of tryptophan and an anthranilic acid has not been previously reported, but
there is literature precedent on the synthesis of such a bond between an indole-nitrogen and
anthranilic acid.6,7 As part of our ongoing synthetic program to examine the pharmacological
properties of macrocyclic peptides,8-12 our attention focused on the synthesis of this
structurally unique family of cyclic peptides.
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Our attention initially focused on the synthesis of the nitrated cyclic peptide psychrophilin C
(3). As far as we know, the cyclization of tripeptides employing a nitro alkylation-based
strategy has no literature precedent. Thus, initial synthetic studies focused on the
intramolecular Mitsunobu-type cyclization of nitroalcohol 9 and intramolecular base-assisted
nitroalkylation of nitrochloride 10 (Scheme 1). Unfortunately, attempts to deliver
psychrophilin C (3) using both of these strategies were unsuccessful. Nitroalcohol 9 was
unreactive towards various azo-dicarboxylate reagents and the use of diisopropyl
azodicarboxylate (DIAD) led to the formation of an azo-dicarboxylate adduct alpha to the
nitro moiety. Treatment of nitrochloride 10 with various bases at room temperature afforded
no reaction and the use of higher temperatures led only to decomposition.
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Scheme 1. Attempted cyclization of precursors 9 and 10 to afford psychrophilin C (3).
In light of these difficulties, and the potent cytotoxic activity of the recently discovered
psychrophilin E (5), we shifted our attention to the synthesis of this alternative target. We
herein present the first synthesis of psychrophilin E (5). The synthesis of 5 provides further
insight into synthetic strategies for the remaining members of the psychrophilin family.
One of the main challenges associated with the synthesis of macrocyclic peptides is tethering
the two ends of the linear precursor. This is due to the need to overcome the ring strain of the
cyclic structure to be formed, which is especially great in the case of tripeptides.13 A study by
Poteau and Trinquier showcased how energetically unfavourable it is to cyclize tri- and
tetrapeptides that contain all cis-amide bonds.14 These considerations highlight the problem
that synthetic chemists face when trying to prepare cyclic tripeptides. Hence, the choice of an
appropriate site to effect the key macrocyclization step is crucial.
Previous work in our laboratory has encountered difficulties involving amide coupling
between the amino group of anthranilic acid residues and an activated carboxyl group.
Therefore, building on our own demonstrated procedure to effect the macrolactamization of a
linear hexapeptide via an N-terminal proline residue,11 psychrophilin E (5) was thus
retrosynthetically disconnected between the proline and tryptophan residues to linear
precursor 11 (Scheme 2). The stereochemistry of the N-acetyl substituent at C-2 in tripeptide
11 was not taken into account at this stage. N-Acetyl benzyl ester 11 was envisioned to be
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accessible by reduction of the nitro group in tripeptide 12. In turn, tripeptide 12 would be
constructed by the addition of benzyl nitroacetate (13) to bromide 14. Tripeptide 15 would
then be assembled via amide coupling of N-Boc-proline (16) and aniline 17, which is
accessible through manipulation of the oxidation states of amide 18. Amide 18 was further
disconnected via the amide bond to afford commercially available compounds 19 and 20.
We also envisioned that deprotection of the benzyl ester and Boc amide groups in tripeptide
12 followed by macrolactamization would provide access to psychrophilin A (1).
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Scheme 2. Retrosynthetic analysis of psychrophilin E (5).
Results and Discussion
We embarked on the proposed synthetic route by coupling indole-3-carboxaldehyde (19) with
2-nitrobenzoic acid (20) using N,N´-dicyclohexylcarbodiimide (DCC) and obtained amide 18
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in 93% yield (Scheme 3). The presence of an aldehyde in amine 19 and an o-nitro group in
carboxylic acid 20 respectively, were required to achieve a successful and high-yielding
amidation. Using reduced and protected variants of 19 and 20 gave lower yields. Reduction
of aldehyde 18 using sodium borohydride followed by reduction of the nitro group using
tin(II) chloride afforded alcohol 21 in 70% yield over two steps. Silyl protection of alcohol 21
then furnished aniline 17 in excellent yield. Subsequent amide coupling of 17 with N-Boc-
proline (16) using DCC followed by silyl deprotection provided alcohol 22 in near
quantitative yield over two steps.
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Scheme 3. Synthesis of alcohol 22.
Attention next turned to nitro alkylation of alcohol 22 to complete the backbone of the
tripeptide (Scheme 4). Alcohol 22 was first converted to the corresponding bromide using
phosphorous tribromide. The crude bromination mixture was then added to a pre-stirred
mixture of benzyl nitroacetate (13), tetra-n-butylammonium bromide and potassium
tert-butoxide in DMSO. Nitro peptide 12 was isolated albeit in 31% yield as a 1:1 epimeric
mixture over two steps. Fortunately, reduction of the nitro group in benzyl ester 12 with zinc
powder and glacial acetic acid in iso-propanol afforded amine 23 in quantitative yield.
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Acetylation of 23 gave N-acetyl tripeptide 11, which underwent near quantitative
hydrogenolysis of the benzyl ester under standard conditions, providing carboxylic acid 24.
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Scheme 4. Synthesis of macrolactamization precursor 24 as a diastereomeric mixture.
With carboxylic acid 24 in hand, attention turned to removal of the N-terminal Boc group by
treatment with TFA and the crude unprotected tripeptide was used directly in the key
cyclization reaction, using our previously optimised macrolactamization conditions (Scheme
5).12 Cyclization was performed under high dilution conditions (0.25 M) by slow addition of
a mixture of the deprotected linear precursor, HATU, and 6-chloro-1-hydroxybenzotriazole
(6Cl-HOBt) in dichloromethane/DMF (1:1) to
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stirred solution of N,N-
diisopropylethylamine (DIPEA) in dichloromethane/DMF (7:1). Pleasingly, psychrophilin E
(5) was isolated in 49% yield, over two steps after stirring at room temperature for 4.5 days.
It was important to leave the reaction for an extended period of time as the isolated yield was
only 30% when the reaction time was 15.5 hours. Although a 1:1 epimeric mixture of linear
precursor 24 was used, surprisingly, only the desired 2S-epimer of macrocycle 5 was
obtained. This observation suggested that only the 2S-epimer of the deprotected linear
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precursor underwent successful macrolactamization. The 2R-epimer of macrocycle 5 was
never isolated, suggesting that the ring strain required to effect the formation of the
macrocyclic system is too high in this case. Theoretical calculations lent support to this
hypothesis, the 2S-epimer of macrocycle 5 was calculated to be more stable than the 2R-
epimer by approximately 30 kJmol-1.15 The Boc-deprotected 2R-epimer of 24 was never
isolated and is speculated that it did not get extracted by ethyl acetate during the aqueous
reaction workup step.
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Scheme 5. Synthesis of psychrophilin E (5) from precursor 24.
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Gratifyingly, the H and 13C NMR spectroscopic data and the HRMS analysis of synthetic
psychrophilin E (5) were in full agreement with those reported for the natural product.4,5 In
addition, the X-ray crystal structure of synthetic 5 matched perfectly with that of naturally
occurring 5 reported by Peng et al.4 Although Ebada et al.5 misassigned the stereochemistry
at C-2 in psychrophilin E as the R configuration, the NMR spectroscopic data recorded in
CDCl3 for our synthetic 5 completely matched the data they reported for the natural product.
The optical rotation value measured for our synthetic material 5 in chloroform ([α]ꢁꢀꢂ +92.0
(c 0.20 in CHCl3)) was 3-fold higher than that reported for the natural product ([α]ꢁꢀꢃ +32.0 (c
0.20 in CHCl3)) by Ebada et al.5 It was noted that a pure sample of synthetic 5 is insoluble in
methanol, therefore we were not able to obtain an accurate optical rotation value to be
compared to that reported by Peng et al. for the natural product ([α]ꢁꢀꢃ +96.0 (c 0.20 in
MeOH)).4 Therefore, based on the spectral data and the single-crystal X-ray structure of our
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synthetic material, the absolute configuration of psychrophilin E (5) is proposed to be
(2S,20S).
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In an attempt to synthesise psychrophilin A (1), nitrobenzyl ester 12 was subjected to
standard hydrogenolysis conditions to effect benzyl ester deprotection. However, this resulted
in decarboxylation and the corresponding nitroalkane product 25 was isolated in 80% yield
Scheme 6). This type of decarboxylation was also observed in our earlier synthetic studies
focused on attempted cyclization of linear psychrophilin precursors via aminolysis of
thioesters.
Scheme 6. Attempted benzyl ester deprotection on nitrobenzyl ester 12.
Although amide coupling between the indole-nitrogen of tryptophan and the carboxylic acid
group of anthranilic acid has not been previously reported, there are examples of direct amide
coupling between the indole-nitrogen of tryptophan and the carboxylic acid group of the
amino acids alanine and leucine, using KF, 18-crown-6 and N,N-diisopropylethylamine
(DIPEA) in MeCN at room temperature.16,17 This observation therefore inspired us to
investigate a more efficient and stereoselective synthetic strategy to access psychrophilin E
(5) based on this approach. In our second generation synthesis, psychrophilin E (5a) was
retrosynthetically disconnected between the proline and tryptophan residues to give linear
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precursor 11a (Scheme 7). Tripeptide 11a in turn, was envisioned to be accessible from
commercially available compounds 16, 26 and 27.
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Scheme 7. Retrosynthesis for second generation synthesis of psychrophilin E (5).
Our revised synthesis commenced by protecting the N-acetyl tryptophan (26) as benzyl ester
28 (Scheme 8). The key amide coupling between the indole-amino group of tryptophan 28
and isatoic anhydride (27) proceeded only at elevated temperature. Pleasingly, the union of
fragments 27 and 28 afforded dipeptide 29 in 53% yield, a moderate yield comparable to
other similar examples reported in the literature.16 Subsequent amide coupling of 29 with N-
Boc-proline (16) using DCC afforded tripeptide 11a, which underwent near quantitative
hydrogenolysis of the benzyl ester under standard conditions, providing carboxylic acid 24a
in 93% yield. Subjection of 24a to the same method used earlier to prepare psychrophilin E
(5) afforded 5 in an isolated yield of 79% over two steps after stirring at room temperature for
3 days. The overall yield of this revised six-step synthesis is 28%, compared to the initial 13-
step synthesis which proceeded with an overall yield of 9%.
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Scheme 8. Synthesis of psychrophilin E (5) from precursor 26.
In summary, the first total synthesis of psychrophilin E (5) has been successfully executed in
six steps with an overall yield of 28%. Highlights of the synthesis include installation of the
rare amide bond between the indole-nitrogen of tryptophan and the anthranilic acid residue,
and macrolactamization of the linear tripeptide to the desired macrocycle 5. The overall
synthetic route is scalable and is anticipated to be amenable to the production of other
members of the psychrophilin family and analogues thereof.
EXPERIMENTAL SECTION
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All reactions were performed under an atmosphere of dry nitrogen in oven dried (100 °C)
glassware unless otherwise stated. Commercially available starting materials and reagents
were used as received unless otherwise noted. All the solvents used were dried by passage
through a column of activated alumina under nitrogen using an LC Technology solvent
purification system. Thin layer chromatography (TLC) was performed using F254 0.2 mm
silica plates, followed by visualisation with UV irradiation at 254 nm, and staining with
ethanolic vanillin solution. Flash column chromatography was performed using 63-100 µm
silica gel. Optical rotations were measured with an automatic polarimeter at 589 nm, and the
concentration was measured in g/100 mL. Infrared (IR) spectra were recorded with a FT-IR
spectrometer using a diamond ATR sampling accessory. NMR spectra were recorded at
ambient temperature as CDCl3 solutions on either a spectrometer operating at 400 MHz for
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1H nuclei and 100 MHz for 13C nuclei or a spectrometer operating at 500 MHz for H nuclei
and 125 MHz for 13C nuclei. All chemical shifts are reported in ppm on the δ scale and were
measured relative to the protium solvent in which the sample was analysed (CDCl3: δ 7.26
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for H NMR and δ 77.0 for 13C NMR). H NMR data is reported as chemical shift in ppm,
followed by multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad or
combination thereof), coupling constants where applicable and relative integral. NMR peak
assignments were confirmed by 2D HSQC and 2D COSY NMR spectra. High-resolution
mass spectra were recorded on a microTOF QII (electrospray ionisation, ESI) mass
spectrometer.
(R)-N-(2-(3-(hydroxymethyl)-1H-indole-1-carbonyl)phenyl)-2-(2-nitroacetamido)pro-
panamide (9). A solution of tetra-n-butylammonium fluoride (2.0 mL of 1.0 M in THF, 2.22
mmol) and glacial acetic acid (0.1 mL, 0.52 mmol) in THF (6 mL) was added to a stirred
solution of TIPS-ether precursor (200 mg, 0.34 mmol) in THF (6 mL) at 0 °C. The reaction
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mixture was stirred for 25 h and allowed to warm to rt The reaction was quenched with sat.
aq. NH4Cl (20 mL) and the mixture extracted with EtOAc (2 × 30 mL). The combined
organic layers were washed with brine (30 mL), dried over MgSO4, filtered and concentrated
in vacuo. Purification of the crude product by flash chromatography (EtOAc) afforded the
title compound 9 (139 mg, 0.33 mmol, 95%) as a white solid; Rf 0.36 (EtOAc); m.p. = 182-
189 °C; [α]ꢁꢀꢄ -42.7 (c 1.00 in DMSO); vmax(neat)/cm-1 3278, 2973, 1680, 1648, 1564, 1535,
1386, 1363, 1332, 1143, 1007, 750, 666; δH (400 MHz; d-DMSO) 10.35 (s, 1 H, NH), 8.77
(d, J = 7.5, 1 H, NH), 8.20 (d, J = 7.9, 1 H, Ar-H), 7.67 (dd, J = 7.1, 1.2, 1 H, Ar-H), 7.65 (td,
J = 7.8, 1.4, 1 H, Ar-H), 7.53 (dd, J = 7.8, 1.4, 1 H, Ar-H), 7.46 (d, J = 7.7, 1 H, Ar-H), 7.40-
7.28 (m, 3 H, 3 × Ar-H), 7.17 (s, 1 H, Ar-H), 5.300, 5.258 (ABq, JAB = 13.6, 2 H, CH2NO2),
5.09 (br t, J = 5.1, 1 H, OH), 4.63 (d, J = 4.0, 2 H, CH2OH), 4.44 (quintet, J = 7.2, 1 H,
CHCH3), 1.06 (d, J = 7.1, 3 H, CHCH3); δC (100 MHz; d-DMSO) 170.4 (CO), 166.3 (CO),
160.8 (CO), 135.9 (C), 135.1 (C), 131.6 (Ar-CH), 129.7 (C), 129.2 (Ar-CH), 128.4 (C), 124.9
(Ar-CH), 124.7 (Ar-CH), 124.5 (Ar-CH), 124.3 (Ar-CH), 123.2 (Ar-CH), 122.2 (C), 119.6
(Ar-CH), 115.9 (Ar-CH), 78.2 (CH2NO2), 55.2 (CH2OH), 48.6 (CHCH3), 17.8 (CHCH3); m/z
(ESI+) [M + Na]+ 447.1286 calcd for C21H20N4NaO6 447.1275.
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1-(2-Nitrobenzoyl)-1H-indole-3-carbaldehyde (18). Indole-3-carboxaldehyde 19 (3.47 g,
23.9 mmol) was added to a stirred solution of 2-nitrobenzoic acid 20 (6.00 g, 35.9 mmol),
N,N´-dicyclohexylcarbodiimide (8.15 g, 39.5 mmol) and 4-dimethylaminopyridine (0.58 g,
4.79 mmol) in THF (120 mL) and the reaction was stirred at rt for 24 h. Formation of a white
precipitate (dicyclohexylurea) was observed, the solid was removed by filtration and the
filtrate concentrated in vacuo. The crude residue was taken up in methanol and the yellow
precipitate formed was collected by filtration. Recrystallization of the yellow solid in
methanol afforded the title compound 18 (6.52 g, 22.2 mmol, 93%) as pale yellow crystals; Rf
0.53 (EtOAc/hexanes 4:1); m.p. = 205-207 °C; vmax(neat)/cm-1 3131, 3072, 2825, 1710, 1672,
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1532, 1347; δH (400 MHz; CDCl3) 10.01 (s, 1 H, CHO), 8.40 (dd, J = 8.3, 1.1, 1 H, Ar-H),
8.30 (dd, J = 7.7, 2.0, 2 H, 2 × Ar-H), 7.916 (td, J = 7.5, 1.1, 1 H, Ar-H), 7.84 (td, J = 8.4,
1.5, 1 H, Ar-H), 7.67 (dd, J = 7.4, 1.5, 1 H, Ar-H), 7.55 (s, 1 H, Ar-H), 7.52-7.45 (m, 2 H, 2 ×
Ar-H); δC (100 MHz; CDCl3) 185.4 (CO), 165.1 (CHO), 145.7 (C), 136.5 (C), 135.1 (Ar-
CH), 134.9 (Ar-CH), 132.0 (Ar-CH), 130.2 (C), 128.9 (Ar-CH), 127.2 (Ar-CH), 126.5 (C),
126.1 (Ar-CH), 125.3 (Ar-CH), 123.4 (Ar-CH), 122.2 (Ar-CH), 116.1 (C); m/z (ESI+) [M +
Na]+ 317.0534 calcd for C16H10N2NaO4 317.0533.
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(2-Aminophenyl)(3-(hydroxymethyl)-1H-indol-1-yl)methanone (21). Sodium borohydride
(431 mg, 11.4 mmol) was added to a stirred solution of nitro-aldehyde 18 (3.05 g, 10.4
mmol) in MeOH/THF 1:1 (40 mL) at 0 °C and the mixture stirred for 5 min at 0 °C. The
reaction was quenched with sat. aq. NH4Cl (15 mL) and the mixture extracted with EtOAc (2
× 15 mL). The combined organic layers were washed with brine (20 mL), dried over MgSO4,
filtered and concentrated in vacuo. Purification of the crude product by flash chromatography
(EtOAc/hexanes 4:1) afforded the intermediate nitro-alcohol as an off-white foam that was
used directly in the next step.
Anhydrous tin(II) chloride (19.6 g, 103 mmol) was added to a stirred solution of nitro-alcohol
in THF (30 mL) at 0 °C. The reaction mixture was stirred for 10 h and allowed to warm to rt.
The reaction was quenched with sat. aq. NaHCO3 (30 mL) and the mixture extracted with
EtOAc (2 × 25 mL). The combined organic layers were washed with brine (30 mL), dried
over MgSO4, filtered and concentrated in vacuo. Purification of the crude product by flash
chromatography (EtOAc/hexanes 3:2) afforded the title compound 21 (1.93 g, 7.26 mmol,
70% over two steps) as a yellow solid; Rf 0.34 (EtOAc/hexanes 4:1); m.p. = 32.0-38.0 °C; v-
max(neat)/cm-1 3471, 3370, 1663, 1613, 1588, 1449, 1350, 1327; δH (400 MHz; CDCl3) 8.27
(dd, J = 8.0, 1.0, 1 H, Ar-H), 7.66 (dd, J = 7.1, 1.2, 1 H, Ar-H), 7.40-7.28 (m, 5 H, 5 × Ar-H),
6.77 (dd, J = 8.6, 0.8, 1 H, Ar-H), 6.73 (td, J = 8.0, 0.9, 1 H, Ar-H), 5.01 (br s, 2 H, NH2),
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4.83 (s, 2 H, CH2), OH not observed; δC (100 MHz; CDCl3) 169.1 (CO), 148.9 (C), 136.7
(C), 133.3 (Ar-CH), 131.2 (Ar-CH), 129.6 (C), 125.7 (Ar-CH), 125.1 (Ar-CH), 123.7 (Ar-
CH), 121.2 (C), 119.3 (Ar-CH), 117.1 (Ar-CH), 116.8 (Ar-CH), 116.4 (Ar-CH), 116.0 (C),
57.3 (CH2); m/z (ESI+) [M + Na]+ 289.0951 calcd for C16H14N2NaO2 289.0947.
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(2-Aminophenyl)(3-(((triisopropylsilyl)oxy)methyl)-1H-indol-1-yl)methanone
(17).
Triisopropylsilyl chloride (2.11 mL, 9.87 mmol) was added to a stirred solution of alcohol 21
(2.19 g, 8.22 mmol) and 2,6-lutidine (1.90 mL, 16.4 mmol) in DMF (15 mL) and the reaction
was stirred at rt for 16 h. The reaction was quenched with aq. lithium bromide (5% w/v, 20
mL) and the mixture extracted with EtOAc (2 × 30 mL). The combined organic layers were
dried over MgSO4, filtered and concentrated in vacuo. Purification of the crude product by
flash chromatography (EtOAc/hexanes 3:2) afforded the title compound 17 (155 mg, 0.37
mmol, 98%) as a yellow oil; Rf 0.73 (EtOAc/hexanes 3:2); vmax(neat)/cm-1 3481, 3381, 2941,
2891, 2864, 1669, 1616, 1590, 1450, 1351, 1329, 1248, 1216, 1126, 1064, 884; δH (400
MHz; CDCl3) 8.30 (d, J = 7.9, 1 H, Ar-H), 7.61 (d, J = 7.9, 1 H, Ar-H), 7.36 (td, J = 7.6, 1.4,
2 H, 2 × Ar-H), 7.32 (s, 1 H, Ar-H), 7.31-7.28 (m, 2 H, 2 × Ar-H), 6.78 (dd, J = 8.5, 0.8, 1 H,
Ar-H), 6.72 (td, J = 8.0, 0.9, 1 H, Ar-H), 5.01 (br s, 2 H, NH2), 4.96 (d, J = 1.3, 2 H, CH2),
1.19-1.12 (m, 3 H, 3 × CH, TIPS), 1.08 (d, J = 2.9, 18 H, 6 × CH3, TIPS); δC (100 MHz;
CDCl3) 169.1 (CO), 148.9 (C), 136.7 (C), 133.1 (Ar-CH), 131.4 (Ar-CH), 129.5 (C), 124.8 (2
× Ar-CH), 123.5 (Ar-CH), 122.3 (C), 119.5 (Ar-CH), 117.0 (Ar-CH), 116.6 (Ar-CH), 116.3
(Ar-CH), 58.5 (CH2), 18.1 (6 × CH3, TIPS), 12.3 (3 × CH, TIPS), one quaternary carbon not
observed but confirmed by mass spectrometry; m/z (ESI+) [M + Na]+ 445.2283 calcd for
C25H34N2NaO2Si 445.2282.
(S)-Tert-butyl
2-((2-(3-(((triisopropylsilyl)oxy)methyl)-1H-indole-1-
solution of N,N´-
carbonyl)phenyl)carbamoyl)pyrrolidine-1-carboxylate (15).
A
dicyclohexylcarbodiimide (685 mg, 3.32 mmol) in CH2Cl2 (60 mL) was added to a stirred
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solution of aniline 17 (936 mg, 2.21 mmol) and Boc-(
L)-Proline 16 (715 mg, 3.32 mmol) in
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CH2Cl2 (40 mL). The reaction mixture was stirred at rt for 19.5 h. The reaction mixture was
filtered through a pad of Celite® and the filtrate was concentrated in vacuo. Purification of the
crude product by flash chromatography (CH2Cl2/EtOAc 9:1) afforded the title compound 15
(1.38 g, 3.21 mmol, 100%) as an off-white foam and as a 1:1.2 mixture of rotameric isomers;
Rf 0.45 (CH2Cl2/EtOAc 9:1); [α]ꢀꢁꢁ –16.1 (c 1.00 in CHCl3); vmax(neat)/cm-1 2940, 2865,
1696, 1666, 1602, 1583, 1520, 1449, 1365, 1352, 1329, 1297, 1255, 1216, 1159, 1124, 1085,
1064, 1031, 1014, 919, 880, 829, 747; δH (400 MHz; CDCl3) 10.09 (br s, 1 H, NH)*, 9.93 (br
s, 1 H, NH), 8.50 (br s, 1 H, Ar-H), 8.37 (d, J = 8.2, 1 H, Ar-H), 7.60-7.51 (m, 3 H, 3 × Ar-
H), 7.39 (t, J = 7.6, 1 H, Ar-H), 7.32 (t, J = 7.4, 1 H, Ar-H), 7.22 (s, 1 H, Ar-H), 7.17 (br s, 1
H, Ar-H), 4.94 (s, 2 H, CH2OTIPS), 4.40 (s, 1 H, CH)*, 4.26 (s, 1 H, CH), 3.59-3.37 (m, 2 H,
CH2, C-3), 2.22 (s, 1 H, H-1a), 2.10 (s, 1 H, H-1b), 1.85 (m, 2 H, CH2, C-2), 1.33 (s, 9 H, 3 ×
CH3, Boc)*, 1.26 (s, 9 H, 3 × CH3, Boc), 1.21-1.12 (m, 3 H, 3 × CH, TIPS), 1.07 (d, J = 6.6,
18 H, 6 × CH3, TIPS); δC (100 MHz; CHCl3) 168.0 (CO), 155.0 (CO), 154.2 (CO), 141.8 (C),
138.4 (C), 136.7 (C), 132.9 (Ar-CH), 130.6 (Ar-CH), 129.6 (C), 125.3 (Ar-CH), 124.1 (2 ×
Ar-CH), 123.0 (Ar-CH), 122.4 (C), 112.1 (Ar-CH), 119.5 (Ar-CH), 116.8 (Ar-CH), 80.4 (C,
Boc), 62.4 (CH)*, 61.6 (CH), 58.4 (CH2OTIPS), 47.1 (CH2, C-3), 29.7 (CH2, C-1), 24.4
(CH2, C-2), 23.8 (CH2, C-2)*, 28.2 (3 × CH3, Boc), 18.1 (6 × CH3, TIPS), 12.0 (3 × CH,
TIPS); m/z (ESI+) [M + Na]+ 642.3350 calcd for C35H49N3NaO5Si 642.3334. * = denotes the
other rotameric isomer.
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(S)-Tert-butyl
2-((2-(3-(hydroxymethyl)-1H-indole-1-carbonyl)phenyl)carbamoyl)-
pyrrolidine-1-carboxylate (22). A solution of tetra-n-butylammonium fluoride (3.5 mL of
1.0M in THF, 3.48 mmol) and glacial acetic acid (0.07 mL, 1.16 mmol) in THF (5 mL) was
added to a stirred solution of TIPS-ether 15 (720 mg, 1.16 mmol) in THF (30 mL) at 0 °C.
The reaction mixture was stirred for 20 h and allowed to warm to rt. The reaction mixture
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was concentrated in vacuo. Purification of the crude product by flash chromatography
(CH2Cl2/EtOAc 7:3) afforded the title compound 22 (540 mg, 1.16 mmol, 100%) as an off-
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white foam and as a 1:1 mixture of rotameric isomers; Rf 0.33 (CH2Cl2/EtOAc 7:3); [α]ꢀꢁꢄ
–
9
135.1 (c 1.00 in CHCl3); vmax(neat)/cm-1 330, 3011, 2923, 2874, 1669, 1602, 1583, 1520,
1450, 1353, 1330, 1253, 1216, 1158, 1121, 1051, 1016, 577, 749; δH (400 MHz; CDCl3) 9.83
(br s, 1 H, NH), 9.42 (br s, 1 H, NH)*, 8.36-8.27 (m, 2 H, 2 × Ar-H), 7.68 (s, 1 H, Ar-H),
7.60-7.48 (m, 2 H, 2 × Ar-H), 7.41-7.32 (m, 2 H, 2 × Ar-H), 7.23 (br s, 2 H, 2 × Ar-H), 4.80
(s, 2 H, CH2OH), 4.34 (br s, 1 H, CH), 4.22 (br s, 1 H, CH)*, 3.35 (br s, 2 H, 2 × H-3), 2.17
(br s, 1 H, H-1a), 2.04 (br s, 1 H, H-1b), 1.79-1.67 (m, 2 H, CH2, H-2), 1.32 (s, 9 H, 3 × CH3,
Boc); δC (100 MHz; CDCl3) 171.8 (CO), 171.4 (CO), 167.8 (CO), 155.4 (C), 154.3 (C)*,
137.7 (C), 137.4 (C)*, 136.5 (C), 132.9 (Ar-CH), 132.8 (Ar-CH)*, 130.1 (Ar-CH), 129.7 (C),
125.6 (Ar-CH), 125.2 (Ar-CH),124.3 (Ar-CH), 123.8 (Ar-CH), 123.5 (C), 122.6 (Ar-CH),
119.6 (Ar-CH), 116.7 (Ar-CH), 80.6 (C, Boc), 62.2 (CH), 61.4 (CH)*, 57.0 (CH2OH), 47.1
(CH2, C-3), 46.8 (CH2, C-3)*, 31.2 (CH2, C-1), 29.4 (CH2, C-1)*, 28.2 (3 × CH3, Boc), 24.4
(CH2, C-2), 23.7 (CH2, C-2)*; m/z (ESI+) [M + Na]+ 486.1986 calcd for C26H29N3NaO5
486.1999. * = denotes the other rotameric isomer.
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Benzyl 2-nitroacetate (13). A solution of N,N´-dicyclohexylcarbodiimide (147 mg, 0.71
mmol) in THF (1 mL) was added to a stirred solution of nitroacetic acid (406 mg, 0.48 mmol)
and benzyl alcohol (0.05 mL, 0.48 mmol) in THF (3 mL) at 0 °C, and then stirred at 0 °C for
50 min. The reaction mixture was filtered through a pad of Celite® and the filtrate was
concentrated in vacuo. Purification of the crude product by flash chromatography (PE/EtOAc
8:2) afforded the title compound 13 (74 mg, 0.48 mmol, 80%) as a yellow wax; Rf 0.40
(PE/EtOAc 8:2); m.p. = 33-36 °C; vmax(neat)/cm-1 3044, 2986, 1748, 1622, 1558, 1496, 1456,
1407, 1379, 1337, 1219, 1179, 1082, 1002, 970, 927, 904, 841, 792, 756, 701, 689; δH (400
MHz; CDCl3) 7.41-7.34 (m, 5 H, 5 × Ar-H), 5.28 (s, 2 H, CH2Ph), 5.18 (s, 2 H, CH2NO2); δC
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(100 MHz; CDCl3) 161.7 (CO), 134.0 (C), 129.1 (Ar-CH), 128.8 (2 × Ar-CH), 128.7 (2 × Ar-
CH), 76.3 (CH2NO2), 68.8 (CH2Ph); m/z (ESI+) [M + Na]+ 218.0428 calcd for C9H9NNaO4
218.0424.
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(2S)-2-((2-(3-(3-(benzyloxy)-2-nitro-3-oxopropyl)-1H-indole-1-
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carbonyl)phenyl)carbamoyl)pyrrolidine-1-carboxylate (12). Phosphorous tribromide (0.3
mL, 2.83 mmol) was added to a stirred solution of alcohol 22 (328 mg, 0.71 mmol) in THF (4
mL) at -78 °C and the mixture stirred for 45 min at 0 °C. The reaction was quenched with sat.
aq. NaHCO3 (10 mL) and the mixture extracted with Et2O (3 × 15 mL). The combined
organic layers were washed with brine (15 mL), dried over MgSO4, filtered and concentrated
in vacuo. The crude bromide product (354 mg) was used directly in the next step without any
purification.
Potassium tert-butoxide (95 mg, 0.85 mmol) was added to a stirred solution of benzyl
nitroacetate (166 mg, 0.85 mmol) and tetra-n-butylammonium bromide (274 mg, 0.85 mmol)
in DMSO (5 mL) and the reaction was stirred at rt for 15 min. Crude bromide in DMSO (1
mL) was added and the reaction was stirred at rt for 2 h. The reaction was quenched with
brine (20 mL) and the mixture extracted with Et2O (3 × 20 mL). The combined organic layers
were dried over MgSO4, filtered and concentrated in vacuo. Purification of the crude product
by flash chromatography (PE/EtOAc 7:3) afforded the title compound 12 (159 mg, 0.25
mmol, 35% over two steps) as a white foam and as a 1:1 mixture of diastereoisomers; Rf 0.46
(PE/EtOAc 3:2); vmax(neat)/cm-1 2972, 2934, 2885, 1752, 1692, 1604, 1583, 1561, 1518,
1488, 1451, 1364, 1328, 1300, 1258, 1216, 1156, 1120, 1088, 1039, 876, 751, 697; δH (400
MHz; CDCl3) 10.03 (d, J = 15.7, 1 H, NH), 9.86 (s, 1 H, NH)*, 8.45 (d, J = 8.3, 1 H, Ar-H),
8.44 (d, J = 8.5, 1 H, Ar-H)*, 8.33 (d, J = 7.8, 1 H, Ar-H), 7.58 (s, 1 H, Ar-H), 7.54 (d, J =
8.7, 1 H, Ar-H), 7.41 (t, J = 8.6, 1 H, Ar-H), 7.37-7.33 (m, 5 H, Ar-H), 7.27-7.25 (m, 2 H,
Ar-H), 7.19 (s, 1 H, Ar-H), 7.15 (s, 1 H, Ar-H), 5.46 (dd, JAX = 9.5, JBX = 5.5, 1 H, CHNO2),
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5.24 (s, 2 H, CH2, Bn), 4.38 (br s, 1 H, CHC-1), 4.24 (br s, 1 H, CHC-1)*, 3.62 (ABX, ΔδAB
= 0.05, JAB = 15.5, JAX = 9.5, JBX = 5.5, 2 H, CH2CHNO2), 3.52 (br s, 1 H, H-3a), 3.36 (br s,
1 H, H-3b), 2.22 (br s, 1 H, H-1a), 2.08 (br s, 1 H, H-1b), 1.85 (br s, 2 H, CH2, H-2), 1.32 (s,
9 H, 3 × CH3, Boc), 1.26 (s, 9 H, 3 × CH3, Boc)*; δC (100 MHz; CDCl3) 172.1 (CO), 171.3
(CO)*, 167.8 (CO), 163.7 (CO), 154.3 (CO), 138.5 (C), 136.3 (C), 134.0 (C), 133.4 (Ar-CH),
133.2 (Ar-CH)*, 130.6 (Ar-CH), 130.3 (Ar-CH)*, 129.5 (C), 129.0 (Ar-CH), 128.8 (Ar-CH),
128.4 (2 × Ar-CH), 126.5 (C), 126.4 (Ar-CH), 125.9 (Ar-CH), 125.5 (Ar-CH), 124.4 (Ar-
CH), 123.3 (Ar-CH), 122.4 (Ar-CH), 122.3 (Ar-CH)*, 118.3 (Ar-CH), 116.9 (Ar-CH), 114.6
(C), 87.2 (CHNO2), 80.5 (C, Boc), 68.8 (CH2, Bn), 62.3 (CHC-1), 61.5 (CHC-1)*, 47.1 (CH2,
C-3), 16.9 (CH2, C-3)*, 31.3 (CH2, C-1), 29.4 (CH2, C-1)*, 28.2 (3 × CH3, Boc), 26.1
(CH2CHNO2), 24.4 (CH2, C-2), 23.8 (CH2, C-2); m/z (ESI+) [M + Na]+ 663.2415 calcd for
C35H36N4NaO8 663.2425. * = denotes the other diastereoisomer.
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Tert-butyl
(2S)-2-((2-(3-(2-amino-3-(benzyloxy)-3-oxopropyl)-1H-indole-1-
carbonyl)phenyl)carbamoyl)pyrrolidine-1-carboxylate (23). Glaciel acetic acid (1 mL,
17.3 mmol) was added to a stirred mixture of nitroester 12 (30 mg, 0.05 mmol) and zinc dust
(18 mg, 0.28mmol) in iso-propanol (2 mL) and the reaction was stirred at rt for 17.5 h. The
reaction was quenched with sat. aq. NaHCO3 (10 mL) and the mixture extracted with EtOAc
(3 × 10 mL). The combined organic layers were washed with brine (20 mL), dried over
MgSO4, filtered and concentrated in vacuo. Purification of the crude product by flash
chromatography (EtOAc/MeOH 95:5) afforded the title compound 23 (28 mg, 0.05 mmol,
100%) as a white foam and as a 1:1 mixture of diastereoisomers; Rf 0.39 (EtOAc/MeOH
95:5); vmax(neat)/cm-1 3357, 2976, 2937, 2885, 1735, 1690, 1604, 1582, 1514, 1492, 1450,
1364, 1330, 1300, 1255, 1214, 1156, 1119, 1088, 1040, 1020, 980, 924, 882, 853, 793, 751,
697; δH (400 MHz; CDCl3) 10.03 (s, 1 H, NH), 9.85 (s, 1 H, NH)*, 8.45 (dd, J = 8.4, 1.6, 1
H, Ar-H), 8.35 (br s, 1 H, Ar-H), 7.58 (d, J = 7.6, 1 H, Ar-H), 7.56 (m, 1 H, Ar-H), 7.39 (t, J
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= 7.4, 2 H, Ar-H), 7.35-7.27 (m, 4 H, 4 × Ar-H), 7.24-7.23 (m, 2 H, 2 × Ar-H), 7.15 (br s, 2
H, Ar-H), 5.16-5.06 (m, 2 H, CH2, Bn), 4.38 (br s, 1 H, CHC-1), 4.24 (br s, 1 H, CHC-1)*,
3.86 (dd, JAX = JBX = 6.3, 1 H, CHNH2), 3.57-3.46 (m, 1 H, H-3a), 3.35 (br s, 1 H, H-3b),
3.07 (ABX, ΔδAB = 0.06, JAB = 14.5, JAX = 5.2, JBX = 7.5, 2 H, CH2CHNH2), 2.21 (br s, 1 H,
H-1a), 2.06 (br s, 1 H, H-1b), 1.83 (br s, 2 H, CH2, H-2), 1.32 (s, 9 H, 3 × CH3, Boc), 1.27 (s,
9 H, 3 × CH3, Boc)*; δC (100 MHz; CDCl3) 174.7 (CO), 172.0 (CO), 171.3 (CO), 167.7
(CO), 138.3 (C), 136.3 (C), 135.4 (C), 133.1 (Ar-CH), 132.9 (Ar-CH)*, 130.9 (C), 130.5 (Ar-
CH), 130.2 (Ar-CH)*, 128.6 (Ar-CH), 128.5 (Ar-CH), 128.2 (Ar-CH), 125.9 (Ar-CH), 125.7
(Ar-CH)*, 125.5 (Ar-CH), 125.2 (Ar-CH)*, 124.1 (Ar-CH), 123.2 (Ar-CH), 122.3 (Ar-CH),
122.3 (Ar-CH), 119.0 (Ar-CH), 118.0 (C), 117.7 (C), 116.8 (Ar-CH), 80.5 (C, Boc), 66.9
(CH2, Bn), 62.3 (CHC-1), 61.5 (CHC-1)*, 54.5 (CHNO2), 47.1 (CH2, C-3), 46.9 (CH2, C-3)*,
31.4 (CH2, C-1), 30.4 (CH2CHNO2), 29.5 (CH2, C-1)*, 28.2 (3 × CH3, Boc), 24.4 (CH2, C-2),
23.8 (CH2, C-2)*; m/z (ESI+) [M + H]+ 611.2848 calcd for C35H39N4O6 611.2864. * =
denotes the other diastereoisomer.
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Tert-butyl
(2S)-2-((2-(3-(2-acetamido-3-(benzyloxy)-3-oxopropyl)-1H-indole-1-
carbonyl)phenyl)carbamoyl)pyrrolidine-1-carboxylate (11). Acetyl chloride (4 µL, 0.06
mmol) was added to a stirred solution of amino ester 23 (31 mg, 0.05 mmol) and
trimethylamine (11 µL, 0.08 mmol) in CH2Cl2 (2 mL) at 0 °C, and the mixture stirred for 2 d
at rt. The reaction was quenched with sat. aq. NaHCO3 (10 mL) and the mixture extracted
with EtOAc (2 × 10 mL). The combined organic layers were washed with brine (20 mL),
dried over MgSO4, filtered and concentrated in vacuo. Purification of the crude product by
flash chromatography (EtOAc/MeOH 95:5) afforded the title compound 11 (28 mg, 0.04
mmol, 85%) as an off-white foam and as a 1:1.2 mixture of diastereoisomers; Rf 0.57
(EtOAc/MeOH 95:5); vmax(neat)/cm-1 2970, 1744, 1691, 1664, 1603, 1583, 1562, 1515, 1451,
1364, 1329, 1297, 1258, 1215, 1157, 1119, 1088, 1019, 919, 875, 794, 750, 698; δH (500
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MHz; CDCl3) 9.93 (s, 1 H, NH)†, 9.86 (s, 1 H, NH), 9.76 (s, 1 H, NH)*, 9.68 (s, 1 H, NH)*†,
8.38-8.30 (m, 2 H, 2 × Ar-H), 7.57 (br s, 1 H, Ar-H), 7.51 (dd, J = 7.7, 4.2, 1 H, Ar-H), 7.40-
7.27 (m, 6 H, 6 × Ar-H), 7.18-7.15 (m, 3 H, 3 × Ar-H), 6.98-6.93 (m, 1 H, Ar-H), 6.28 (br s,
1 H, NH), 6.22 (br s, 1 H, NH)*, 5.12-4.95 (m, 3 H, CH2, Bn and CHNHAc), 4.36 (br s, 1 H,
CHC-1), 4.24 (br s, 1 H, CHC-1)*, 3.50-3.33 (m, 2 H, CH2, H-3), 3.23 (ABX, ꢀδAB = 0.03,
JAB = 14.5, JAX = 5.0, JBX = 5.1, 2 H, CH2CHNHAc), 2.18 (br s, 1 H, H-1a), 2.01 (br s, 1 H,
H-1b), 1.952 (s, 3 H, CH3, Ac), 1.947 (s, 3 H, CH3, Ac)*, 1.81-1.77 (m, 2 H, CH2, H-2), 1.32
(br s, 9 H, 3 × CH3, Boc), 1.30 (br s, 9 H, 3 × CH3, Boc)*; δC (125 MHz; CDCl3) 172.1 (CO),
171.4 (CO), 169.9 (CO), 169.8 (CO)*, 167.6 (CO), 167.6 (CO)*, 155.4 (CO), 138.0 (C),
137.8 (C), 136.11 (C), 136.07 (C), 133.0 (Ar-CH), 132.8 (Ar-CH)*, 131.0 (2 × C), 130.3 (Ar-
CH), 130.0 (Ar-CH), 128.6 (2 × Ar-CH), 128.2 (2 × Ar-CH), 125.9 (Ar-CH), 125.5 (Ar-CH),
125.2 (Ar-CH)*, 124.2 (Ar-CH), 123.6 (Ar-CH), 123.4 (Ar-CH)*, 123.0 (Ar-CH), 122.8 (Ar-
CH)*, 118.9 (Ar-CH), 116.6 (Ar-CH), 80.5 (C, Boc), 67.4 (CH2, Bn), 62.2 (CHC-1), 61.4
(CHC-1)*, 52.5 (CHNHAc), 47.1 (CH2, C-3), 31.3 (CH2, C-1), 28.4 (CH2, C-1)*, 28.2 (3 ×
CH3, Boc), 27.3 (CH2CHNHAc), 27.2 (CH2CHNHAc)*, 24.4 (CH2, C-2), 23.8 (CH2, C-2)*,
23.2 (CH3, Ac), 23.1 (CH3, Ac)*; m/z (ESI+) [M + Na]+ 675.2780 calcd for C37H40N4NaO7
675.2789. * = denotes the other diastereoisomer. † = denotes the other rotameric isomer.
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Nα-Acetyl-1-(2-((S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)benzoyl)-
tryptophan (24). 10% Pd/C (10 wt. %, 10 mg, 0.01 mmol) was added to a stirred solution of
benzyl ester 11 (70 mg, 0.11 mmol) in MeOH (8 mL) at rt under N2 atmosphere, and the
mixture stirred under H2 atmosphere for 2.3 h. The reaction mixture was filtered through a
pad of Celite® and the filtrate was concentrated in vacuo. Purification of the crude product by
flash chromatography (10% MeOH/CH2Cl2) afforded the title compound 24 (60 mg, 0.11
mmol, 99%) as a white foam and as a 1:1.4 mixture of diastereoisomers; Rf 0.06
(CH2Cl2/MeOH 9:1); vmax(neat)/cm-1 3303, 2979, 2934, 2885, 1659, 1604, 1584, 1522, 1482,
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1452, 1366, 1331, 1254, 1216, 1159, 1124, 1040, 920, 876, 752; δH (400 MHz; CDCl3) 9.63
(s, 1 H, NH), 9.53 (s, 1 H, NH)*, 8.37-8.03 (m, 2 H, 2 × Ar-H), 7.57 (d, J = 7.4, 2 H, 2 × Ar-
H), 7.50 (br s, 1 H, Ar-H), 7.35 (t, J = 7.4, 1 H, Ar-H), 7.30 (t, J = 7.4, 1 H, Ar-H), 7.22 (br s,
1 H, Ar-H), 7.05-7.00 (m, 1 H, Ar-H), 6.60 (br s, 1 H, NH), 4.81 (dd, J = 12.5, 5.5, 1 H,
CHNHAc), 4.25 (br s, 1 H, CHC-1), 3.47 (br s, 2 H, CH2, H-3)*, 3.39 (br s, 2 H, CH2, H-3),
3.19 (ABX, ꢀδAB = 0.04, JAB = 14.8, JAX = 5.6, JBX = 5.3, 2 H, CH2CHNHAc), 2.08 (br s, 2
H, CH2, H-1), 1.93 (s, 3 H, CH3, Ac)*, 1.92 (s, 3 H, CH3, Ac), 1.80 (br s, 2 H, CH2, H-2),
1.31 (br s, 9 H, 3 × CH3, Boc)*, 1.29 (br s, 9 H, 3 × CH3, Boc); δC (100 MHz; CDCl3) 173.3
(CO), 173.2 (CO), 170.8 (CO), 167.63 (CO), 167.61 (CO), 137.0 (C), 135.9 (2 × C), 132.6
(Ar-CH), 131.2 (C), 130.0 (Ar-CH), 129.9 (Ar-CH), 125.9 (Ar-CH), 125.2 (Ar-CH), 124.3
(Ar-CH), 123.1 (Ar-CH), 119.1 (Ar-CH), 117.2 (C), 116.6 (Ar-CH), 81.1 (C, Boc), 62.0
(CHC-1)*, 61.3 (CHC-1), 52.7 (CHNHAc), 47.2 (CH2, C-3), 46.9 (CH2, C-3)*, 31.2 (CH2,
C-1)*, 29.7 (CH2, C-1), 28.22 (3 × CH3, Boc), 28.19 (3 × CH3, Boc)*, 27.0 (CH2CHNHAc),
24.3 (CH2, C-2), 23.5 (CH2, C-2)*, 23.0 (CH3, Ac)*, 22.9 (CH3, Ac); m/z (ESI+) [M + Na]+
585.2303 calcd for C30H34N4NaO7 585.2320.
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* = denotes the other diastereoisomer.
Tert-butyl
(S)-2-((2-(3-(2-nitroethyl)-1H-indole-1-carbonyl)phenyl)carbamoyl)-
pyrrolidine-1-carboxylate (25). 10% Pd/C (10 wt. %, 10 mg, 0.01 mmol) was added to a
stirred solution of tripeptide benzyl ester precursor (19 mg, 0.03 mmol) in MeOH (3 mL) at rt
under N2 atmosphere, and the mixture stirred under H2 atmosphere for 2 h. The reaction
mixture was filtered through a pad of Celite® and the filtrate was concentrated in vacuo.
Purification of the crude product by flash chromatography (PE/EtOAc 3:2) afforded the title
compound 25 (12 mg, 0.02 mmol, 80%) as an off-white foam and as a 1:1.3 mixture of
rotameric isomers; Rf 0.44 (PE/EtOAc 3:2); [α]ꢁꢀꢅ –27.8 (c 1.00 in CHCl3); vmax(neat)/cm-1
3353, 2962, 2920, 2853, 1689, 1670, 1597, 1583, 1549, 1528, 1451, 1366, 1328, 1259, 1219,
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1151, 1127, 1088, 928, 857, 751, 681; δH (500 MHz; d-DMSO) 10.08 (br s, 1 H, NH), 8.17
(d, J = 7.8, 1 H, Ar-H)*, 8.12 (d, J = 7.2, 1 H, Ar-H), 7.71 (d, J = 6.7, 1 H, Ar-H), 7.65 (t, J =
7.7, 1 H, Ar-H), 7.53 (d, J = 7.5, 1 H, Ar-H)*, 7.50 (d, J = 7.7, 1 H, Ar-H), 7.44 (d, J = 7.5, 1
H, Ar-H), 7.37-7.30 (m, 3 H, 3 × Ar-H), 7.18 (s, 1 H, Ar-H), 4.86 (t, J = 7.1, 2 H, CH2NO2),
4.16-4.12 (m, 1 H, CHC-1)*, 4.08 (dd, J = 8.6, 4.1, 1 H, CHC-1), 3.32 (t, J = 7.1, 2 H,
CH2CH2NO2), 3.25-3.19 (m, 1 H, H-3a), 3.18-3.13 (m, 1 H, H-3b), 1.91-1.83 (m, 1 H, H-1a),
1.56-1.48 (m, 1 H, H-1b), 1.45-1.38 (m, 2 H, 2 × H-2), 1.32 (s, 9 H, 3 × CH3, Boc)*, 1.28 (s,
9 H, 3 × CH3, Boc); δC (125 MHz; d-DMSO) 172.0 (CO), 166.8 (CO), 166.7 (CO), 136.0
(C), 135.9 (C), 132.3 (Ar-CH), 130.5 (C), 129.9 (Ar-CH), 128.6 (C), 126.1 (Ar-CH), 125.3
(Ar-CH), 125.2 (Ar-CH), 125.1 (C), 124.7 (Ar-CH), 124.3 (Ar-CH)*, 123.9 (Ar-CH), 119.5
(Ar-CH), 116.4 (Ar-CH), 79.0 (C, Boc), 75.0 (CH2, CH2NO2), 60.3 (CHC-1)*, 60.1 (CHC-1),
47.0 (CH2, C-3)*, 46.8 (CH2, C-3), 30.6 (CH2, C1), 28.5 (3 × CH3, Boc)*, 28.4 (3 × CH3,
Boc), 23.2 (CH2, C-2), 22.6 (CH2CH2NO2); m/z (ESI+) [M + Na]+ 529.2069 calcd for
C27H30N4NaO6 529.2058. * = denotes the minor rotameric isomer
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Nα-Acetyl-(
L
)-tryptophan benzyl ester (28). Benzyl bromide (0.4 mL, 3.65 mmol) was
-tryptophan (26) (600 mg, 2.44 mmol) and DIPEA (1.1
added to a stirred solution of N-Ac-(
L
)
mL, 6.09 mmol) in DMF (30 mL) under N2 atmosphere, and reaction mixture was stirred for
23 h. The reaction was diluted by the addition of water (60 mL) and the mixture extracted
with EtOAc (3 × 50 mL). The combined organic layers were dried over MgSO4, filtered and
concentrated in vacuo. Purification of the crude product by flash chromatography (EtOAc)
afforded the title compound 28 (814 mg, 2.44 mmol, 99%) as a white foam; Rf 0.46 (EtOAc);
[α]ꢁꢀꢃ –13.5 (c 1.00 in CHCl3); vmax(neat)/cm-1 3399, 3275, 3059, 2930, 1732, 1652, 1518,
1456, 1340, 1175, 1130, 1009, 739, 696; δH (500 MHz; CDCl3) 8.44 (s, 1 H, NH, indole),
7.50 (d, J = 7.9, 1 H, Ar-H), 7.33-7.29 (m, 4 H, 4 × Ar-H), 7.23-7.21 (m, 2 H, 2 × Ar-H), 7.16
(td, J = 6.8, 1.0, 1 H, Ar-H), 7.08 (td, J = 7.6, 1.1, 1 H, Ar-H), 6.70 (d, J = 2.4, 1 H, Ar-H),
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6.14 (d, J = 7.9, 1 H, NH), 5.09, 5.05 (ABq, J = 12.2, 2 H, CH2, Bn), 5.00-4.96 (m, 1 H,
CHNHAc), 3.29 (ABX, ΔδAB = 0.03, JAB = 14.7, JAX = 5.5, JBX = 5.3, 2 H, CH2CHNHAc),
1.89 (s, 3 H, CH3, Ac); δC (125 MHz; CDCl3) 172.0 (CO), 170.1 (CO), 136.2 (C), 135.3 (C),
128.6 (2 × Ar-CH), 128.5 (3 × Ar-CH), 127.8 (C), 123.0 (Ar-CH), 122.2 (Ar-CH), 119.6 (Ar-
CH), 118.5 (Ar-CH), 111.4 (Ar-CH), 109.6 (C), 67.2 (CH2, Bn), 53.3 (CH), 27.6
(CH2CHNHAc), 23.2 (CH3, Ac); m/z (ESI+) [M + Na]+ 359.1370 calcd for C20H20N2NaO3
359.1366.
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Nα-Acetyl-1-(2-aminobenzoyl)-(
L)-tryptophan benzyl ester (29). Isatoic anhydride (27)
(508 mg, 3.12 mmol), 18-crown-6 (2.06 g, 7.79 mmol), potassium fluoride (453 mg,
7.79 mmol) and DIPEA (0.54 mL, 3.12 mmol) were added to a solution of indole 28 (524
mg, 1.56 mmol) in MeCN (50 mL) at rt, and the reaction mixture was stirred for 21 h at 60
°C. The reaction was quenched with water (60 mL) and the mixture extracted with EtOAc (3
× 60 mL). The combined organic layers were washed with brine (60 mL), dried over MgSO4,
filtered and concentrated in vacuo. Purification of the crude product by flash chromatography
(EtOAc/PE 7:3) afforded the title compound 29 (369 mg, 0.83 mmol, 53%) as a white foam;
Rf 0.61 (EtOAc/PE 8:2); [α]ꢀꢁꢂ –3.0 (c 1.00 in CHCl3); vmax(neat)/cm-1 3473, 3355, 3061,
2925, 1738, 1656, 1617, 1589, 1535, 1490, 1451, 1364, 1327, 1248, 1215, 1181, 1162, 1127,
1029, 915, 885, 818, 787, 746, 697, 667; δH (500 MHz; CDCl3) 8.25 (d, J = 8.0, 1 H, Ar-H),
7.35 (td, J = 7.0, 1.1, 1 H, Ar-H), 7.32-7.22 (m, 6 H, 6 × Ar-H), 7.16-7.14 (m, 2 H, 2 × Ar-
H), 7.06 (s, 1 H, Ar-H), 6.77 (d, J = 8.5, 1 H, Ar-H), 6.70 (t, J = 7.8, 1 H, Ar-H), 6.14 (d, J =
7.6, 1 H, NHAc), 5.10-4.96 (m, 5 H, CH2, Bn; Ar-NH2 and CHNHAc), 3.24 (ABX, ΔδAB
=
0.03, JAB = 14.7, JAX = 5.6, JBX = 4.8, 2 H, CH2CHNHAc), 1.95 (s, 3 H, CH3, Ac); δC (125
MHz; CDCl3) 171.5 (CO), 169.7 (CO), 168.8 (CO), 149.0 (C), 136.1 (C), 134.8 (C), 133.4
(Ar-CH), 131.3 (Ar-CH), 131.0 (C), 128.65 (2 × Ar-CH), 128.61 (Ar-CH), 128.3 (Ar-CH),
126.1 (Ar-CH), 125.0 (Ar-CH), 123.7 (Ar-CH), 118.8 (Ar-CH), 117.1 (Ar-CH), 117.1 (Ar-
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CH), 116.7 (Ar-CH), 116.2 (Ar-CH), 116.0 (C), 115.6 (C), 67.5 (CH2, Bn), 52.8 (CHNHAc),
27.4 (CH2CHNHAc), 23.2 (CH3, Ac); m/z (ESI+) [M + Na]+ 478.1751 calcd for
C27H25N3NaO4 478.1737.
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Tert-butyl
carbonyl)phenyl)carbamoyl)pyrrolidine-1-carboxylate (11a).
dicyclohexylcarbodiimide (251 mg, 1.21 mmol) in CH2Cl2 (14 mL) was added to a stirred
solution of aniline 29 (369 mg, 0.81 mmol) and Boc-( )-proline 16 (261 mg, 1.21 mmol) in
(S)-2-((2-(3-((S)-2-acetamido-3-(benzyloxy)-3-oxopropyl)-1H-indole-1-
A
solution of N,N´-
L
CH2Cl2 (14 mL). After stirring for 30 min at rt, the reaction mixture was filtered through a
pad of Celite® and the filtrate was concentrated in vacuo. Purification of the crude product by
flash chromatography (EtOAc/PE 7:3) afforded the title compound 11a (381 mg, 0.58 mmol,
72%) as an off-white foam and as a 1:1.2 mixture of rotameric isomers; Rf 0.28 (EtOAc/PE
7:3); vmax(neat)/cm-1 3302, 3062, 3011, 2931, 2855, 1738, 1657, 1518, 1457, 1439, 1371,
1341, 1213, 1190, 1130, 1010, 741, 696, 666; [α]ꢁꢀꢄ –6.7 (c 1.00 in CHCl3); δH (500 MHz;
CDCl3) 9.93 (br s, 1 H, NH), 9.76 (br s, 1 H, NH)*, 8.38 (d, J = 7.7, 1 H, Ar-H), 8.29 (br s, 1
H, Ar-H), 7.57 (br s, 1 H, Ar-H), 7.52 (d, J = 7.7, 1 H, Ar-H), 7.38 (t, J = 7.6, 1 H, Ar-H),
7.35-7.26 (m, 5 H, 5 × Ar-H), 7.17-7.15 (m, 3 H, 3 × Ar-H), 6.98-6.93 (m, 1 H, Ar-H), 6.17
(br s, 1 H, NH), 5.11, 5.018 (ABq, J = 12.3, 2 H, CH2, Bn), 4.99-4.95 (m, 1 H, CHNHAc),
4.36 (br s, 1 H, CHC-1), 4.24 (br s, 1 H, CHC-1)*, 3.50 (br s, 1 H, H-3a), 3.35 (br s, 1 H, H-
3b), 3.28-3.17 (m, 2 H, CH2CHNHAc), 2.19 (br s, 1 H, H-1a), 2.04 (br s, 1 H, H-1b), 1.95 (s,
3 H, CH3, Ac), 1.82 (br s, 2 H, 2 × H-2), 1.33 (s, 9 H, 3 × CH3, Boc), 1.28 (s, 9 H, 3 × CH3,
Boc)*; δC (125 MHz; CDCl3) 171.4 (CO), 169.8 (CO), 167.6 (CO), 136.1 (CO), 134.8 (CO),
133.4 (Ar-CH), 132.8 (Ar-CH), 131.0 (C), 130.5 (Ar-CH), 130.0 (Ar-CH), 128.6 (Ar-CH),
128.2 (Ar-CH), 125.8 (Ar-CH), 125.5 (Ar-CH), 125.2 (Ar-CH), 124.2 (Ar-CH), 123.4 (Ar-
CH), 122.8 (Ar-CH), 118.9 (Ar-CH), 116.6 (Ar-CH), 80.5 (C, Boc), 67.5 (CH2, Bn), 52.5
(CHNHAc), 47.1 (CH2, C-3), 29.4 (CH2, C-1), 28.2 (3 × CH3, Boc), 27.3 (CH2CHNHAc),
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24.4 (CH2, C-2), 23.8 (CH2, C-2)*, 23.2 (CH3, Ac), four quaternary carbons not observed but
confirmed by mass spectrometry; m/z (ESI+) [M + Na]+ 675.2803 calcd for C37H40N4NaO7
675.2789. * = denotes the minor rotameric isomer.
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Nα-Acetyl-1-(2-((S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)benzoyl)-
L-
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tryptophan (24a). 10% Pd/C (10 wt. %, 20 mg, 0.02 mmol) was added to a stirred solution
of benzyl ester 11a (83 mg, 0.13 mmol) in MeOH (15 mL) at rt under N2 atmosphere, and the
mixture stirred under H2 atmosphere for 2 h. The reaction mixture was filtered through a pad
of Celite® and the filtrate was concentrated in vacuo. Purification of the crude product by
flash chromatography (10% MeOH/CH2Cl2) afforded the title compound 24a (67 mg, 0.12
mmol, 93%) as a white foam and as a 1:1.5 mixture of rotameric isomers; Rf 0.06
(CH2Cl2/MeOH 9:1); [α]ꢁꢀꢄ –28.2 (c 1.00 in CHCl3); vmax(neat)/cm-1 3303, 2979, 2934, 2885,
1659, 1604, 1584, 1522, 1482, 1452, 1366, 1331, 1254, 1216, 1159, 1124, 1040, 920, 876,
752; δH (400 MHz; d-DMSO) 12.78 (s, 1 H, CO2H), 10.10 (s, 1 H, NH), 8.21-8.14 (m, 2 H,
Ac-NH, Ar-H), 7.67-7.63 (m, 2 H, 2 × Ar-H), 7.56-7.45 (m, 2 H, 2 × Ar-H), 7.36-7.30 (m, 3
H, 3 × Ar-H), 7.14 (s, 1 H, Ar-H), 4.48-4.43 (m, 1 H, CHNHAc), 4.16 (d, J = 8.3, 1 H, CHC-
1)*, 4.11 (d, J = 8.5, 1 H, CHC-1), 3.33-3.24 (m, 2 H, CH2, H-3), 3.17-2.90 (m, 2 H,
CH2CHNHAc), 1.92-1.86 (m, 1 H, H-1a), 1.77 (s, 3 H, CH3, Ac), 1.56-1.41 (m, 3 H, CH2, H-
2, H-1b), 1.33 (s, 9 H, 3 × CH3, Boc)* 1.30 (s, 9 H, 3 × CH3, Boc); δC (100 MHz; d-DMSO)
173.1 (CO), 171.4 (CO), 170.9 (CO)*, 169.2 (CO), 166.3(CO)*, 166.2 (CO), 153.5 (CO)*,
153.0 (CO), 135.7 (C)*, 135.6 (C), 135.4 (C), 131.6 (Ar-CH), 130.5 (C), 129.3 (Ar-CH),
128.3 (C), 126.0 (Ar-CH), 124.7 (Ar-CH), 124.5 (Ar-CH), 124.2 (Ar-CH), 123.4 (Ar-CH),
118.8 (Ar-CH), 116.6 (C), 115.8 (Ar-CH), 18.7 (C, Boc)*, 78.5 (C, Boc), 59.8 (CHC-1), 59.6
(CHC-1)*, 51.8 (CHNHAc), 46.5 (CH2, C-3)*, 46.3 (CH2, C-3), 30.1 (CH2, C-1), 29.1 (CH2,
C-1)*, 28.0 (3 × CH3, Boc)*, 27.9 (3 × CH3, Boc), 26.4 (CH2CHNHAc), 23.4 (CH2, C-2)*,
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22.7 (CH2, C-2), 22.3 (CH3, Ac); m/z (ESI+) [M + Na]+ 585.2303 calcd for C30H34N4NaO7
585.2320. * = denotes the minor rotameric isomer.
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Psychrophilin E (5). Method A: TFA (0.8 mL, 10 mmol) was added to a stirred solution of
Boc-amide 24 (26 mg, 46 µmol) in CH2Cl2 (5.2 mL) at 0 °C, and the mixture stirred at rt for
30 min. The reaction mixture was concentrated in vacuo, and then put under high vacuum for
30 min to remove the excess TFA. The crude TFA salt (30 mg) was used directly in the next
step without further purification.
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Crude amine-TFA salt in CH2Cl2 (10.5 mL) was added to a mixture of HATU (53 mg, 138
µmol) and 6Cl-HOBt (19 mg, 115 µmol) in CH2Cl2 (10.5 mL) and DMF (21 mL). This
activated linear peptide mixture was then transferred into two separate syringes, one of each
was added dropwise (0.4 mL/h) using a syringe pump into two separate solutions of DIPEA
(1.3 mL, 7.5 mmol) in CH2Cl2 (26 mL) and DMF (4 mL). The reaction mixtures were
combined after stirring at rt for 4 d and then quenched with H2O (100 mL) and the mixture
extracted with EtOAc (4 × 80 mL). The combined organic layers were washed with brine
(100 mL), dried over MgSO4, filtered and concentrated in vacuo. Purification of the crude
product by flash chromatography (CH2Cl2/MeOH 19:1) afforded psychrophilin E (5) (10 mg,
49%, 22.5 µmol) as a white powder; Rf 0.44 (CH2Cl2/MeOH 95:5); [α]ꢁꢀꢂ +92.0 (c 0.20 in
CHCl3); vmax(neat)/cm-1 3351, 2924, 2853, 1698, 1665, 1623, 1604, 1523, 1486, 1450, 1366,
1332, 1285, 1251, 1224, 1172, 1153, 1120, 1104, 1058, 1038, 943, 895, 878, 843, 771; δH
(500 MHz; CDCl3) 8.63 (d, J = 8.4, 1 H, Ar-H), 8.52 (s, 1 H, NH-Abz), 7.83 (dd, J = 8.4, 1.2,
1 H, Ar-H), 7.68 (d, J = 7.7, 1 H, Ar-H), 7.58-7.56 (m, 2 H, 2 × Ar-H), 7.44 (td, J = 7.7, 1.1,
1 H, Ar-H), 7.39 (td, J = 6.7, 2.2, 2 H, 2 × Ar-H), 6.85 (s, 1 H, Ar-H), 6.21 (d, J = 8.0, 1 H,
NH-Ac), 5.10 (ddd, J = 11.5, 8.1, 5.5, 1 H, H-2), 4.56 (dd, J = 8.3, 2.9, 1 H, H-20), 3.37 (dd,
J = 12.8, 5.4, 1 H, H-3a), 3.25 (ddd, JA = 7.2, JB = JC = 9.4, 1 H, H-23a), 2.87 (t, J = 12.1, 1
H, H-3b), 2.17 (ddd, JA = 9.8, JB = 8.1, JC = 5.3, 1 H, H-23b), 2.09-2.05 (m, 1 H, H-21a),
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2.06 (s, 3 H, CH3, H-25), 1.70-1.67 (m, 1 H, H-21b), 1.54-1.50 (m, 1 H, H-22a), 1.24-1.20
(m, 1 H, H-22b); δC (125 MHz; CDCl3) 172.3 (CO), 169.9 (CO), 167.4 (CO), 166.2 (CO),
135.5 (C), 133.4 (C), 132.6 (Ar-CH), 131.8 (Ar-CH), 129.7 (C), 128.2 (C), 126.4 (Ar-CH),
126.1 (Ar-CH), 125.6 (Ar-CH), 124.6 (Ar-CH), 123.8 (Ar-CH), 118.3 (Ar-CH), 117.2 (Ar-
CH), 116.4 (C), 59.8 (CH, C-20), 51.0 (CH, C-2), 47.7 (CH2, C-23), 29.8 (CH2, C-3), 26.3
(CH2, C-21), 24.7 (CH2, C-22), 23.1 (CH3, C-25); m/z (ESI+) [M + H]+ 445.1879 calcd for
C25H25N4O4 445.1870.
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Method B: TFA (0.7 mL, 9.4 mmol) was added to a stirred solution of Boc-amide 24a (24
mg, 43 µmol) in CH2Cl2 (4.8 mL) at 0 °C, and the mixture stirred at rt for 1 h. The reaction
mixture was concentrated in vacuo, and then put under high vacuum for 30 min to remove the
excess TFA. The crude TFA salt (24 mg) was used directly in the next step without further
purification.
Crude amine-TFA salt in CH2Cl2 (9.5 mL) was added to a mixture of HATU (49 mg, 128
µmol) and 6Cl-HOBt (18 mg, 107 µmol) in CH2Cl2 (9.5 mL) and DMF (19 mL). This
activated linear peptide mixture was then added dropwise (0.4 mL/h) using a syringe pump to
a solution of DIPEA (2.4 mL, 13.8 mmol) in CH2Cl2 (48 mL) and DMF (7 mL). After stirring
at rt for 6 d, the reaction mixture was quenched with H2O (150 mL) and the mixture extracted
with EtOAc (4 × 80 mL). The combined organic layers were washed with brine (150 mL),
dried over MgSO4, filtered and concentrated in vacuo. Purification of the crude product by
flash chromatography (CH2Cl2/MeOH 19:1) afforded psychrophilin E (5) (15 mg, 79%, 34
µmol) as a white powder. The spectroscopic data was in agreement with that reported above.
Supporting Information
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1H and 13C spectra for all new compounds and synthetic psychrophilin E, X-ray
crystallographic analysis of synthetic psychrophilin E, and theoretical methods used for
computational modelling of 2S-5 and 2R-5.
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