Key words
proline - conformation - switchable - amides - peptides
Proline’s unique cyclic structure combined with the resulting conformational restraints
enables this amino acid to play an important role in the folding and stability of
many proteins and peptides.[1]
[2]
[3]
[4] In addition, when incorporated in a peptide, proline is the only protein amino acid
featuring a tertiary amide, which basically suggests that the cis as well as the trans rotamer of the amide should be observed.[5]
[6]
[7] Astonishingly, only very few prolines (ca. 10%) incorporated in native peptides
and proteins do actually prefer the cis conformation of their peptidyl–CO–N-prolyl bond.[7] Synthetic studies on how to influence the stability and the folding properties of
peptides and proteins have so far mainly been carried out with 3- and 4-substituted
proline derivatives 1–4,[8] in which the additional substituents mainly exert strong effects on the conformation
of the pyrrolidine ring (Figure [1], grey background). Among the broad variety of 3- and 4-substituted prolines,[9]
[10] the 4-substituted derivatives 2–4 represent the more frequent approach and such compounds have often been employed
to tune the properties of collagen[11] and other biomolecules.[12]
Figure 1 Examples of 3-, 4- and 5-substituted proline derivatives
In contrast to the conformational effects on the pyrrolidine core observed for prolines
1–4, an additional substituent(s) at the 5-position of proline, as in compounds 5–7, can be useful to impact the conformation of the CO–N-prolyl bond.[13]
[14]
[15]
[16] A cis to trans ratio of 66:34 was observed for the (5S)-tert-butyl derivative 5,[17] whilst 5,5-dimethyl substitution, as in proline 6, led to an even higher prevalence of the cis rotamer of 90% in water.[18]
[19]
[20] Further studies including the incorporation of 5,5-dimethylproline in peptides demonstrated
the high potential of such derivatives in biological and medicinal applications.[21] In a recent study on the influence of positive charges on the conformation of amide
bonds, it was found that protonated 5-aminomethylprolines 7a represent a valuable alternative to known 5-substituted proline derivatives, favoring
a cis conformation of the prolyl amide.[22]
[23] Besides a strong conformational fixation observed for 7a in a range of solvents (87% to >95% cis rotamer), the charge-induced effect is pH-dependent and it can thus be used to reversibly
favor either the cis rotamer 7a or the trans rotamer 7b through the addition of acid or base.[22] Against the background that these initial studies on the conformational fixation
of prolylamide 7a were carried out with racemic 2,5-trans-configured proline derivatives, we now describe stereoselective syntheses of trans-configured 5-aminomethylprolines.[24] The suitability of the newly obtained Fmoc-protected acids for incorporation into
peptides is further demonstrated with the synthesis of a Fmoc-l-Val-(2S,5S)amPro-OMe dipeptide.
The chiral, enantiomerically pure pyrrolidines 8a and 8b used as starting materials (Scheme [1]) were readily available from adipic acid dichloride via dibromination, esterification
and cyclization with (S)-1-phenylethylamine.[25] Following a procedure reported by Yamamoto,[25b] the separation of the diastereoisomers 8a and 8b was achieved through a combination of crystallization and column chromatography.
Scheme 1 Synthesis of Fmoc-protected 5-(dimethylaminomethyl)prolines 13a and 13b; brsm = based on recovered starting material
The selective monohydrolysis of 8a has been reported in the literature using 1.7 equivalents of sodium hydroxide in
a methanol/water mixture at 20 °C to give 9a in 79% yield after three days.[26] In this work, the single hydrolysis could be achieved in a much shorter reaction
time of 4.5 hours with 1.5 equivalents of sodium hydroxide at a slightly elevated
temperature of 45 °C. Unreacted 8a was separated from 9a by column chromatography or, more conveniently for larger scale reactions, via stepwise
extraction at accurately defined pH values. N,N′-Dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) were chosen for
the synthesis of amide 10a from acid 9a and dimethylamine due to the later on simple removal of N,N′-dicyclohexylurea through crystallization (Scheme [1]). The purification of amide 10a was possible via column chromatography or extraction, which is again favorable for
larger reaction scales.
The subsequent change of the protecting group from (S)-α-methylbenzyl to benzyl was necessary since the originally intended reduction of
the amide in 10a with borane–dimethyl sulfide[27] led to a number of side reactions including demethylation at the amino group. In
various attempts, suitable conditions for selective reduction of 10a could neither be found with the borane–dimethyl sulfide nor with the borane–THF adduct.[28] The (S)-α-methylbenzyl group of 10a was thus cleaved through hydrogenation with palladium on charcoal (1 bar H2), and alkylation with benzyl bromide gave 11a in high yield without purification of the intermediate (Scheme [1]).
Overall, amide 11a could be obtained in three steps from 9a (83%), requiring no chromatography and only one crystallization step to remove N,N′-dicyclohexylurea from 10a.
The selective reduction of the amide in 11a was then achieved with borane–dimethyl sulfide under strictly anhydrous conditions.[27] Borane species coordinated to the aminoalkylproline 12a were removed through heating in refluxing methanol (Scheme [1]).[27b]
[c]
[29] In this particular step, complete consumption of the starting material 11a was crucial since the separation of 12a from unreacted starting material 11a through column chromatography turned out to be laborious.
Deprotection of 12a and attachment of the 9-fluorenylmethoxycarbonyl (Fmoc) group was conducted in three
steps. First, the cleavage of the benzyl protecting group was achieved via hydrogenation
with palladium on charcoal in the presence of trifluoroacetic acid. Secondly, the
ester moiety was hydrolyzed with 10 equivalents of sodium hydroxide in aqueous methanol,
and the reaction mixture was then neutralized and concentrated in vacuo. In the third
step, attachment of the Fmoc protecting group was performed under slightly alkaline
conditions using sodium bicarbonate in aqueous tetrahydrofuran (Scheme [1]). After quenching with trifluoroacetic acid, the Fmoc-protected proline 13a could be purified by HPLC. From reactions on larger scales, 13a was obtained in good purity only by purification through extraction (see the Supporting
Information for NMR spectra). After completion of the synthesis of 13a, the synthetic strategy was transferred to the preparation of 13b from the (2R,5R)-configured diester 8b. In this sequence, a clean reduction of the amide 11a to 12a again required a prior change of the protecting group to benzyl. The yields for the
single reaction steps were in most cases similar to those reached in the preparation
of 13a from 8a, thus indicating the reproducibility of the sequence. The lower outcome for the final
steps from 11b to 13b can be attributed to the smaller reaction scale which complicates especially the
borane reduction. Determination of the optical purity of the target compounds 13a and 13b by chiral HPLC gave ee values of more than 95%.
To enable further synthetic options, and to allow conformational fixation as a result
of acid-induced deprotection, a N-Boc protected 5-aminomethylproline derivative was prepared (Scheme [2]). Starting from acid 9a, coupling with ammonia provided amide 14a. The exchange of the chiral auxiliary for the benzyl protecting group proceeded as
described for 10a (see Scheme [1]). Only the benzylation step required a longer reaction time to give 15a. Selective reduction of the amide functionality, removal of complexed borane, and
subsequent Boc protection gave 16a. In the next steps, the benzyl protecting group was cleaved by hydrogenolysis and
the ester was hydrolyzed. As the protection with Fmoc chloride led to an unexpected
twofold attachment of the Fmoc group including the formation of a mixed anhydride
from the carboxylic acid, this final transformation was combined with a mild alkaline
hydrolysis to provide 17a. After the final step, the orthogonally protected proline derivative was purified
via extraction to give 17a in 95% purity according to 1H NMR analysis.[30]
Scheme 2 Synthesis of N-Boc-aminomethylproline 17a
Scheme 3 Synthesis of protonated Fmoc-l-Val-(2S,5S)amPro-OMe (19)
Finally, the applicability of the new building blocks for peptide synthesis was demonstrated
by coupling the N-Boc-aminomethylproline methyl ester 18 to N-Fmoc-protected valine. The building block 18 was obtained in quantitative yield via hydrogenation of 16a. Coupling of 18 and Fmoc-l-Val-OH could be achieved using HATU {1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate} and N,N-diisopropylethylamine (DIPEA) in dry DMF. HPLC monitoring of the reaction course
indicated a conversion of 18 to the dipeptide of 66% without formation of detectable side products (Scheme [3]). Work-up and purification by column chromatography finally gave the dipeptide 19; during this step cleavage of the Boc group occurred since a polar solvent mixture
and twofold chromatography on silica had to be used for purification. The fact that
19 showed only one set of signals when analyzed by 1H and 13C NMR under acidic conditions demonstrates that the conformational fixation is also
effective for this particular type of N-prolyl dipeptide.
In summary, the synthesis of three new 5-(aminomethyl)proline building blocks has
been achieved from literature-known precursors. The eight to nine step sequences could
be performed with good to high yields for all single steps. Suitability for larger
reaction scales is shown since only two purifications by column chromatography are
required within each synthetic pathway. The starting materials 8a and 8b were available in two steps. All three 5-(aminomethyl)prolines were prepared in Fmoc-protected
form to allow direct use in peptide synthesis, which has been demonstrated through
incorporation of 18 into the Fmoc-Val-amPro-OMe dipeptide 19.
Solvents and reagents were obtained from commercial sources and used as received.
Analytical TLC was carried out on Merck silica gel plates using shortwave (254 nm)
UV light, KMnO4 [3 g KMnO4, 20 g potassium carbonate, 5 mL aqueous sodium hydroxide (5% w/w) in 300 mL H2O] or ninhydrin (200 mg ninhydrin in 100 mL ethanol) to visualize components. Merck
silica gel (40–63 μm) was used for flash column chromatography. Yields were calculated
in percent (%) based on the employed starting material or in percent based on recovered
starting material (% brsm). Compounds 8a and 8b, which were used as starting materials, were prepared according to ref. 25b. The
analytical data obtained were in agreement with those reported in the above reference.
Melting points were recorded using a MPM-H2 instrument. Optical rotations were obtained
using a Perkin–Elmer 241 polarimeter. IR spectra were recorded as a substance film
on a sodium chloride crystal using a Jasco FT/IR 410 spectrometer. 1H and 13C NMR spectra were recorded on Bruker Avance 600 (1H: 600 MHz, 13C: 151 MHz), Bruker Avance 360 (1H: 360 MHz, 13C: 91 MHz) and Bruker Avance 400 (1H: 400 MHz, 13C: 101 MHz) spectrometers. Chemical shifts are reported in parts per million (ppm)
and coupling constants (J) are reported in Hertz (Hz). 1H NMR measurements are referenced to TMS (0 ppm) or the signals for the residual protons
of the used solvent: CDCl3 (7.26 ppm), CD3CN (1.94 ppm) or D2O (4.79 ppm). For 13C NMR measurements, CDCl3 and CD3CN were used as solvents using CHCl3 (77.0 ppm) or CD3CN (1.32 ppm) as standards. The following abbreviations are used for the description
of signals: s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet) and
br s (broad signal). Mass spectra were recorded using electron impact (EI) or electrospray
ionization (ESI). A JOEL GC mate II GC-MS-System, Bruker Daltonik microOTOF II, and
Bruker Daltonik maXis 4G were used for HRMS measurements. High-performance liquid
chromatography (HPLC) was performed on a Varian 940 C8 column (21.2 × 150 mm × 5 μm)
with PDA (photodiode array) detection (solvent A: 0.1% CF3CO2H in water; solvent B: acetonitrile; flow rate: 20 mL/min). HPLC-MS was performed
using an Agilent 1100 HPLC-MS with a Zorbax XDB-C8 Column (2.1 × 50 mm × 2.6 μm) with
PDA detection at 254 nm and online ESI mass spectrometry detection (solvent A: 0.1%
CF3CO2H in water; solvent B: acetonitrile containing 0.1% CF3CO2H; flow rate: 0.5 mL/min. Method A: gradient A/B: 90%/10% → 10%/90% over 20 min; method
B: gradient A/B: 100%/0% → 10%/90% acetonitrile over 24 min). Chiral HPLC was performed
using a Chiralpak IC column (4.6 × 250 mm × 5 μm) with UV detection at 254 nm (method
A: solvent A: hexane; solvent B: ethanol containing 0.1% ethylenediamine; flow rate:
0.7 mL/min, A/B: 80%/20% isocratic; method B: solvent A: hexane; solvent B: isopropanol
containing 0.1% ethylenediamine; flow rate: 0.7 mL/min, A/B: 80%/20% isocratic).
(2S,5S)-5-(Methoxycarbonyl)-1-[(S)-1-phenylethyl]pyrrolidine-2-carboxylic Acid (9a)
(2S,5S)-5-(Methoxycarbonyl)-1-[(S)-1-phenylethyl]pyrrolidine-2-carboxylic Acid (9a)
Sodium hydroxide (2 M, 14.3 mL) was added to 8a (5.53 g, 19.0 mmol) in water (58 mL) and methanol (143 mL) and was stirred for 4.5
h at 45 °C. Water (35 mL) was added and the mixture was extracted with ethyl acetate
(1 × 200 mL then 1 × 75 mL). The combined organic phases were dried over sodium sulfate
and the solvent was removed under reduced pressure. Column chromatography (silica
gel, hexane/ethyl acetate = 4:1 → 100% ethyl acetate → ethyl acetate/methanol = 9:1)
allowed recovery of unreacted 8a (710 mg, 2.45 mmol). The aqueous phase of the previous extraction was adjusted to
a pH value of 3 with hydrochloric acid (3 M), supersaturated with sodium chloride
and extracted with ethyl acetate (4 × 75 mL). The combined organic phases were dried
over sodium sulfate and the solvent was removed under reduced pressure to give title
compound 9a (4.43 g, 16.0 mmol, 84%, 97% brsm) as a white solid.
Mp 107.4 °C; Rf
= 0.7 (ethyl acetate) [UV, KMnO4]; [α]24
589 –110.7 (c 0.5, CHCl3).
IR (NaCl): 2977, 2956, 2881, 1733, 1456, 1374, 1310, 1207, 1166, 767, 704 cm–1.
1H NMR (360 MHz, CDCl3): δ = 7.36–7.22 (m, 5 H), 4.20 (q, J = 6.7 Hz, 1 H), 3.92 (dd, J = 1.3 Hz, J = 11.0 Hz, 1 H), 3.63 (d, J = 7.3 Hz, 1 H), 3.57 (s, 3 H), 2.66–2.51 (m, 1 H), 2.16–2.02 (m, 2 H), 1.88–1.80
(m, 1 H), 1.38 (d, J = 6.8 Hz, 3 H).
13C NMR (91 MHz, CDCl3): δ = 176.1, 172.8, 142.5, 128.8, 128.0, 127.1, 63.9, 63.4, 60.1, 51.6, 29.8, 28.8,
22.9.
HRMS (ESI): m/z [M + H]+ calcd for C15H20NO4: 278.1387; found: 278.1391.
(2R,5R)-5-(Methoxycarbonyl)-1-[(S)-1-phenylethyl]pyrrolidine-2-carboxylic Acid (9b)
(2R,5R)-5-(Methoxycarbonyl)-1-[(S)-1-phenylethyl]pyrrolidine-2-carboxylic Acid (9b)
For the synthesis of the (2R,5R)-isomer 9b, starting material 8b (413 mg, 1.42 mmol) was treated as described above to give the title compound 9b (390 mg, 1.40 mmol, 98%, quant. brsm) as a white solid.
Rf
= 0.7 (ethyl acetate) [UV, KMnO4]; [α]24
589 +10.8 (c 0.5, CHCl3).
IR (NaCl): 2974, 2952, 1734, 1617, 1456, 1436, 1375, 1306, 1282, 1200, 1166, 1060,
763, 702 cm–1.
1H NMR (360 MHz, CDCl3): δ = 7.33 (d, J = 7.2 Hz, 2 H), 7.29 (t, J = 7.4 Hz, 2 H), 7.25 (t, J = 7.2 Hz, 1 H), 4.10 (d, J = 5.7 Hz, 2 H), 3.87 (d, J = 9.1 Hz, 1 H), 3.60 (s, 3 H), 2.52–2.45 (m, 1 H), 2.28–2.21 (m, 1 H), 1.93–1.89
(m, 2 H), 1.46 (d, J = 6.4 Hz, 3 H).
13C NMR (91 MHz, CDCl3): δ = 175.8, 173.6, 141.8, 128.5, 128.4, 128.3, 63.6, 63.4, 60.0, 51.7, 29.4, 29.1,
21.3.
HRMS (ESI): m/z [M + Na]+ calcd for C15H19NNaO4: 300.1206; found: 300.1210.
Methyl (2S,5S)-5-(Dimethylcarbamoyl)-1-[(S)-1-phenylethyl]pyrrolidine-2-carboxylate (10a)
Methyl (2S,5S)-5-(Dimethylcarbamoyl)-1-[(S)-1-phenylethyl]pyrrolidine-2-carboxylate (10a)
Diisopropylethylamine (1.00 mL, 0.75 g, 5.82 mmol), 1-hydroxybenzotriazole (0.79 g,
5.82 mmol) and N,N′-dicyclohexylcarbodiimide (1.64 g, 7.94 mmol) were added to a solution of monoester
9a (1.47 g, 5.29 mmol) in dry chloroform (20.7 mL) under an argon atmosphere. Dimethylamine
(2 M in dry tetrahydrofuran, 5.29 mL, 10.6 mmol) was added dropwise and the resulting
mixture was stirred for 16 h overnight at room temperature. Subsequently, water (90
mL) and a saturated aqueous solution of sodium carbonate (10 mL) were added and the
mixture was extracted with chloroform (4 × 40 mL). The combined organic phases were
washed with a saturated aqueous solution of sodium chloride and dried over sodium
sulfate. The solvent was removed under reduced pressure. The crude mixture was redissolved
in ethyl acetate and kept at 4 °C overnight. After removal of the white precipitate
by filtration (containing mainly N,N′-dicyclohexylurea) and washing of the filter cake with small amounts of ethyl acetate,
the filtrate was concentrated under reduced pressure. The crude product was further
purified by column chromatography (silica gel, hexane/ethyl acetate = 2:1 → 1:1).
The title compound 10a (1.37 g, 4.52 mmol, 85%) was obtained as a white solid.
Mp 69.1 °C; Rf
= 0.2 (1:1 hexane/ethyl acetate) [UV, KMnO4]; [α]24
589 –86.9 (c 0.5, CHCl3).
IR (NaCl): 2949, 2359, 2355, 1750, 1730, 1645, 1494, 1455, 1259, 1192, 1152, 1057,
705 cm–1.
1H NMR (600 MHz, CDCl3): δ = 7.38–7.34 (m, 2 H), 7.29 (t, J = 7.6 Hz, 2 H), 7.21 (t, J = 7.4 Hz, 1 H), 4.07–3.93 (m, 3 H), 3.73 (s, 3 H), 2.81 (s, 3 H), 2.63–2.55 (m, 1 H),
2.35 (s, 3 H), 2.24–2.15 (m, 1 H), 1.87–1.80 (m, 1 H), 1.65 (dd, J = 7.9 Hz, J = 12.1 Hz, 1 H), 1.26 (d, J = 4.5 Hz, 3 H).
13C NMR (151 MHz, CDCl3): δ = 177.7, 173.8, 145.3, 128.2, 127.7, 127.1, 63.3, 60.5, 60.1, 51.7, 26.5, 35.4,
29.9, 28.5, 23.4.
HRMS (ESI): m/z [M + H]+ calcd for C17H25N2O3: 305.1860; found: 305.1857.
Methyl (2R,5R)-5-(Dimethylcarbamoyl)-1-[(S)-1-phenylethyl]pyrrolidine-2-carboxylate (10b)
Methyl (2R,5R)-5-(Dimethylcarbamoyl)-1-[(S)-1-phenylethyl]pyrrolidine-2-carboxylate (10b)
For the synthesis of the (2R,5R)-isomer 10b, starting material 9b (356 g, 1.30 mmol) was treated as described above to give the title compound 10b (287 mg, 0.94 mmol, 73%) as a white solid.
Mp 111.3 °C; Rf
= 0.4 (ethyl acetate) [UV, KMnO4]; [α]24
589 +42.6 (c 0.5, CHCl3).
IR (NaCl): 3559, 3463, 2970, 2949, 1734, 1647, 1494, 1455, 1434, 1398, 1366, 1320,
1280, 1195, 1167, 1130, 1090, 1059, 767, 703 cm–1.
1H NMR (600 MHz, CDCl3): δ = 7.35 (d, J = 6.6 Hz, 2 H), 7.26 (t, J = 7.6 Hz, 2 H), 7.18 (t, J = 7.2 Hz, 1 H), 4.35 (d, J = 5.4 Hz, 1 H), 4.16 (d, J = 5.2 Hz, 1 H), 3.99 (d, J = 8.4 Hz, 1 H), 3.36 (s, 3 H), 2.83 (s, 3 H), 2.79 (s, 3 H), 2.52–2.41 (m, 1 H),
1.80–1.71 (m, 2 H), 1.33 (d, J = 5.7 Hz, 3 H).
13C NMR (91 MHz, CDCl3): δ = 175.6, 173.6, 143.0, 128.0, 127.4, 126.8, 63.7, 59.8, 59.5, 50.7, 36.4, 35.0,
28.9, 28.4, 22.0.
HRMS (EI): m/z [M + Na]+ calcd for C17H24N2NaO3: 327.1680; found: 327.1672.
Methyl (2S,5S)-1-Benzyl-5-(dimethylcarbamoyl)pyrrolidine-2-carboxylate (11a)
Methyl (2S,5S)-1-Benzyl-5-(dimethylcarbamoyl)pyrrolidine-2-carboxylate (11a)
A mixture of 10a (609 mg, 2.00 mmol), palladium on carbon (10% w/w, 120 mg, 0.11 mmol) and trifluoroacetic
acid (3.90 mL) in dry ethyl acetate (39 mL) was stirred under a hydrogen atmosphere
(1 bar) at 50 °C for 2 h. The reaction course was monitored via TLC. When the reaction
was finished, the mixture was filtered over Celite® and the filter cake was washed with ethyl acetate. The solvent was removed under
reduced pressure and the crude product was dissolved in acetonitrile (19.5 mL). Potassium
carbonate (1.35 g, 9.77 mmol) and benzyl bromide (0.70 mL, 1.01 g, 5.89 mmol) were
added and the reaction mixture was stirred for 2 h. Subsequently, water (40 mL) was
added and the aqueous layer was extracted with ethyl acetate (4 × 40 mL). The combined
organic phases were washed with a saturated aqueous solution of sodium chloride and
dried over sodium sulfate. The solvent was removed under reduced pressure and the
crude product was purified via column chromatography (silica, hexane/ethyl acetate = 1:1)
to give the title compound 11a (527 mg, 1.82 mmol, 91%) as a colorless oil.
Rf
= 0.3 (hexane/ethyl acetate = 1:2) [UV, KMnO4]; [α]24
589 –84.5 (c 0.5, CHCl3).
IR (NaCl): 2952, 2850, 1733, 1645, 1495, 1454, 1399, 1265, 1200, 1127, 748, 702 cm–1.
1H NMR (600 MHz, CDCl3): δ = 7.36 (d, J = 7.2 Hz, 2 H), 7.29 (t, J = 7.2 Hz, 2 H), 7.22 (t, J = 7.2 Hz, 1 H), 4.13–3.94 (m, 3 H), 3.78 (d, J = 12.5 Hz, 1 H), 3.67 (s, 3 H), 2.89 (s, 3 H), 2.70 (s, 3 H), 2.42 (qd, J = 9.3 Hz, J = 12.4 Hz, 1 H), 2.30–2.20 (m, 1 H), 1.99 (t, J = 9.1 Hz, 1 H), 1.78 (tdd, J = 2.8 Hz, J = 9.4 Hz, J = 12.1 Hz, 1 H).
13C NMR (151 MHz, CDCl3): δ = 175.3, 173.3, 139.0, 129.1, 128.1, 127.1, 63.9, 59.9, 53.9, 51.6, 36.8, 35.5,
28.4, 28.1.
HRMS (EI): m/z [M + H]+ calcd for C16H23N2O3: 291.1703; found: 291.1697.
The 1H NMR and 13C NMR data are in agreement with those previously reported for the racemic compound.[22]
Methyl (2R,5R)-1-Benzyl-5-(dimethylcarbamoyl)pyrrolidine-2-carboxylate (11b)
Methyl (2R,5R)-1-Benzyl-5-(dimethylcarbamoyl)pyrrolidine-2-carboxylate (11b)
For the synthesis of the (2R,5R)-isomer 11b, starting material 10b (243 mg, 0.80 mmol) was treated as described above to give the title compound 11b (209 mg, 0.72 mmol, 90%) as a colorless oil. The spectroscopic data obtained are
in agreement with the data described above.
[α]24
589 +90.0 (c 0.5, CHCl3).
Methyl (2S,5S)-1-Benzyl-5-[(dimethylamino)methyl]pyrrolidine-2-carboxylate (12a)
Methyl (2S,5S)-1-Benzyl-5-[(dimethylamino)methyl]pyrrolidine-2-carboxylate (12a)
Borane dimethyl sulfide (2 M in dry tetrahydrofuran, 4.00 mL) was added to a solution
of amide 11a (801 mg, 2.76 mmol) in dry tetrahydrofuran (40 mL) under an argon atmosphere and
the reaction mixture was stirred for 6 h at 50 °C. Subsequently, dry methanol (35
mL) was added carefully and the mixture was stirred at 75 °C for 18 h. The reaction
course was monitored by TLC. After the reaction was finished, the solvent was removed
under reduced pressure and the crude product was purified via column chromatography
(silica gel, deactivated with triethylamine, hexane/ethyl acetate = 1:1). Title compound
12a (485 mg, 1.66 mmol, 60%) was obtained as a clear viscous oil.
Rf
= 0.1 (hexane/ethyl acetate = 1:2) [UV, KMnO4]; [α]24
589 –164.2 (c 0.5, CHCl3).
IR (NaCl): 2948, 2817, 2764, 2364, 1732, 1454, 1196, 1159, 1036, 996, 845, 745, 700
cm–1.
1H NMR (360 MHz, CDCl3, TFA salt): δ = 7.32–7.18 (m, 5 H), 4.07 (d, J = 13.7 Hz, 1 H), 3.74 (d, J = 13.7 Hz, 1 H), 3.64 (s, 3 H), 3.59 (d, J = 7.9 Hz, 1 H), 3.40–3.33 (m, 1 H), 2.36 (dd, J = 3.8 Hz, J = 12.2 Hz, 1 H), 2.30–2.17 (m, 8 H), 2.12–2.00 (m, 1 H), 1.83–1.73 (m, 2 H).
13C NMR (151 MHz, CDCl3, TFA salt): δ = 174.7, 139.8, 128.5, 128.2, 126.9, 65.2, 63.0, 59.6, 53.3, 51.0,
46.2, 28.9, 27.9.
HRMS (EI): m/z [M + H]+ calcd for C16H25N2O2: 277.1911; found: 277.1909.
The 1H NMR and 13C NMR data are in agreement with those previously reported for the racemic compound.[22]
Methyl (2R,5R)-1-Benzyl-5-[(dimethylamino)methyl]pyrrolidine-2-carboxylate (12b)
Methyl (2R,5R)-1-Benzyl-5-[(dimethylamino)methyl]pyrrolidine-2-carboxylate (12b)
For the synthesis of the (2R,5R)-isomer 12b, starting material 11b (174.2 mg, 0.60 mmol) was treated as described above to give the title compound 12b (81.7 mg, 0.29 mmol, 49%) as a colorless oil. The spectroscopic data obtained are
in agreement with the data described above.
[α]24
589 +166.0 (c 0.5, CHCl3).
(2S,5S)-1-{[(9H-Fluoren-9-yl)methoxy]carbonyl}-5-[(dimethylammonio)methyl]pyrrolidine-2-carboxylate
(13a)
(2S,5S)-1-{[(9H-Fluoren-9-yl)methoxy]carbonyl}-5-[(dimethylammonio)methyl]pyrrolidine-2-carboxylate
(13a)
A mixture of amine 12a (276 mg, 1.00 mmol), palladium on carbon (10% w/w, 100 mg, 0.1 mmol) and trifluoroacetic
acid (0.46 mL, 0.68 mg, 6.00 mmol) in dry ethyl acetate (9.5 mL) was stirred for 2.5
h at 50 °C under a hydrogen atmosphere (1 bar). The reaction course was monitored
by TLC. When the reaction was finished, the mixture was filtered over glass wool and
the filter bed was washed with ethyl acetate. The solvent was removed under reduced
pressure and the obtained residue was dissolved in methanol (36 mL). After addition
of sodium hydroxide (2 M, 5.60 mL), the mixture was stirred for 1 h at 30 °C. The
pH value was adjusted to a value of 7 and the solvent was removed under reduced pressure.
The obtained residue was dissolved in acetonitrile and the remaining solid was filtered
off. The filter cake was washed with acetonitrile and the solvent was removed under
reduced pressure. The residue was dissolved in tetrahydrofuran (21 mL) and water (6
mL). Sodium bicarbonate (168 mg, 2.00 mmol) and 9-fluorenylmethoxycarbonyl chloride
(388 mg, 1.50 mmol) were added and the mixture was stirred for 2.5 h at 30 °C. Trifluoroacetic
acid (1 mL) was added and the mixture was extracted with ethyl acetate (1 × 20 mL).
After addition of hexane (30 mL), the organic phase was washed with water (4 × 30
mL). The combined aqueous phases were extracted with ethyl acetate (7 × 50 mL) and
the completion of the extraction was monitored by TLC. The solvent was concentrated
under reduced pressure and trifluoroacetic acid (1 mL) was added. After washing with
water, the solvent was removed under reduced pressure to give the title compound 13a (186 mg, 0.47 mmol, 47%) as a white solid. The compound was further purified via
HPLC (gradient A/B: min 0–5: 90%/10%; min 5–7: 90%/10% → 60%/40%; min 7–21: 60%/40%
→ 50%/50%; min 21–22: 50%/50% → 10%/90%; min 22–28: 10%/90%; min 28–29: 10%/90% →
90%/10%; min 29–33: 90%/10%; t
R = 16.3 min).
Rf
= 0.5 (acetonitrile + 1% TFA) [UV, ninhydrin]; [α]24
589 –43.9 (c 0.5, CHCl3).
IR (NaCl): 3059, 2961, 2891, 2713, 2607, 2501, 1699, 1451, 1415, 1343, 1268, 1198,
1131, 761, 740, 721 cm–1.
1H NMR (600 MHz, CD3CN, TFA salt): δ = 7.87–7.80 (m, 2.0 H), 7.67–7.60 (m, 2.0 H), 7.47–7.40 (m, 2.0 H),
7.40–7.32 (m, 2.0 H), 4.85 (dd, J = 4.9 Hz, J = 11.3 Hz, 0.1 H), 4.69 (dd, J = 4.5 Hz, J = 11.3 Hz, 0.1 H), 4.44 (dd, J = 6.4 Hz, J = 10.6 Hz, 0.9 H), 4.40–4.36 (m, 1.8 H), 4.32 (dd,
J = 1.8 Hz, J = 8.3 Hz, 0.9 H), 4.24 (t, J = 6.6 Hz, 1.0 H), 4.15 (d,
J
= 8.3 Hz, 0.1 H), 3.91–3.87 (m, 0.1 H), 3.26 (ddd, J = 1.8 Hz,
J = 9.8 Hz, J = 13.2 Hz, 0.9 H), 3.07 (ddd, J = 2.2 Hz, J = 8.5 Hz, J = 13.5 Hz, 0.9 H), 2.96 (d, J = 5.1 Hz, 2.7 H), 2.82 (d, J = 5.1 Hz, 2.7 H), 2.76–2.72 (m, 0.1 H), 2.58–2.54 (m, 0.1 H), 2.52 (d, J = 5.1 Hz, 0.3 H), 2.38 (d,
J = 5.1 Hz, 0.3 H), 2.32–2.12 (m, 2.0 H), 2.08–2.00 (m, 1.0 H), 1.70–1.66 (m, 1.0 H).
13C NMR (151 MHz, CDCl3, TFA salt): δ = 173.2, 158.4, 154.9, 144.9, 144.6, 142.3, 142.2, 128.9, 128.8, 128.5,
128.4, 128.3, 128.2, 126.1, 125.9, 125.7, 121.11, 121.08, 69.4, 67.0, 65.0, 61.0,
60.6, 60.4, 55.1, 53.6, 48.3, 47.8, 46.1, 45.1, 43.8, 43.7, 29.2, 28.7, 28.0, 27.6.
HRMS (ESI): m/z [M + H]+ calcd for C23H27N2O4: 395.1965; found: 395.1961.
HPLC-MS (ESI): Method A, t
R = 6.71 min, m/z = 395 [M + H]+; chiral HPLC: Method A, t
R = 14.79 min.
(2R,5R)-1-{[(9H-Fluoren-9-yl)methoxy]carbonyl}-5-[(dimethylammonio)methyl]pyrrolidine-2-carboxylate
(13b)
(2R,5R)-1-{[(9H-Fluoren-9-yl)methoxy]carbonyl}-5-[(dimethylammonio)methyl]pyrrolidine-2-carboxylate
(13b)
For the synthesis of (2R,5R)-isomer 13b, starting material 12b (50.9 mg, 0.18 mmol) was treated as described above to give the title compound 13b (27.7 mg, 0.07 mmol, 39%) as a white solid. The spectroscopic data obtained are in
agreement with the data described above.
[α]24
589 +47.3 (c 0.25, CHCl3).
HPLC-MS (ESI): Method A, t
R = 6.89 min, m/z = 395 [M + H]+; chiral HPLC: Method A, t
R = 25.93 min.
Methyl (2S,5S)-5-Carbamoyl-1-[(S)-1-phenylethyl]pyrrolidine-2-carboxylate (14a)
Methyl (2S,5S)-5-Carbamoyl-1-[(S)-1-phenylethyl]pyrrolidine-2-carboxylate (14a)
Diisopropylethylamine (11.5 mL, 8.71 g, 67.4 mmol), 1-hydroxybenzotriazole (2.19 g,
16.2 mmol) and N,N′-dicyclohexylcarbodiimide (5.57 g, 27.0 mmol) were added to a solution of monoester
9a (3.74 g, 13.5 mmol) in dry chloroform (54 mL) under an argon atmosphere. Ammonium
chloride (2.16 g, 40.4 mmol) was added and the mixture was stirred for 4.5 h at room
temperature. Subsequently, hexane (200 mL) was added and the mixture was extracted
with hydrochloric acid (3 M, 5 × 100 mL). The combined aqueous phases were washed
with hexane (100 mL) and the pH of the aqueous phase was carefully adjusted to a value
of 9 by addition of potassium carbonate under vigorous stirring. The aqueous phase
was extracted with ethyl acetate (5 × 200 mL), washed with a saturated aqueous solution
of sodium chloride and dried over sodium sulfate. The solvent was removed under reduced
pressure and the title compound 14a was obtained as a white solid which was used in the next step without further purification.
Mp 183.3 °C; Rf
= 0.3 (ethyl acetate) [UV, KMnO4]; [α]24
589 –129.1 (c 0.5, CHCl3).
IR (NaCl): 3458, 3235, 3179, 2992, 1730, 1684, 1666, 1454, 1436, 1391, 1376, 1202,
1162, 1140, 991, 766, 702, 612 cm–1.
1H NMR (360 MHz, CDCl3): δ = 7.34–7.20 (m, 5 H), 7.06 (br s, 1 H), 5.73 (br s, 1 H), 4.09 (q, J = 6.7 Hz, 1 H), 3.78 (dd, J = 1.9 Hz, J = 11.0 Hz, 1 H), 3.56 (d, J = 7.6 Hz, 1 H), 3.54 (s, 3 H), 2.57 (ddt, J = 7.3 Hz, J = 11.1 Hz, J = 13.0 Hz, 1 H), 2.08 (tt, J = 7.7 Hz, J = 12.7 Hz, 1 H), 1.96 (ddd, J = 2.0 Hz, J = 7.8 Hz, J = 12.8 Hz, 1 H), 1.78 (dd, J = 7.6 Hz, J = 12.8 Hz, 1 H), 1.36 (d, J = 6.7 Hz, 3 H).
13C NMR (91 MHz, CDCl3): δ = 179.9, 173.5, 143.9, 128.6, 127.6, 127.2, 64.5, 64.2, 60.4, 51.2, 30.4, 28.8,
23.5.
HRMS (ESI): m/z [M + Na]+ calcd for C15H20N2NaO3: 299.1366; found: 299.1369.
Methyl (2S,5S)-1-Benzyl-5-carbamoylpyrrolidine-2-carboxylate (15a)
Methyl (2S,5S)-1-Benzyl-5-carbamoylpyrrolidine-2-carboxylate (15a)
A mixture of 14a (max. 13.5 mmol), palladium on carbon (10% w/w, 450 mg, 0.42 mmol) and trifluoroacetic
acid (4.50 mL) in dry ethyl acetate (45 mL) was stirred for 2 h at 45 °C under a hydrogen
atmosphere. The reaction course was monitored by TLC. When the reaction was finished
the mixture was filtered over Celite® and washed with ethyl acetate. The solvent was removed under reduced pressure and
the obtained residue was dissolved in acetonitrile (67.5 mL). Potassium carbonate
(14.9 g, 108 mmol) and benzyl bromide (8.02 mL, 11.5 g, 67.5 mmol) were added and
the mixture was stirred at 45 °C for 16 h. Subsequently, hexane (150 mL) was added
and the mixture was extracted with water (5 × 75 mL). The combined aqueous phases
were washed with hexane (20 mL) and the pH was adjusted to a value of 11 by addition
of potassium carbonate under vigorous stirring. The aqueous phase was extracted with
ethyl acetate (5 × 250 mL), dried over sodium sulfate and the solvent was removed
under reduced pressure. The title compound 15a (2.59 g, 9.46 mmol, 70% from 9a) was obtained as a white solid and was used for the next step without further purification.
Mp 136.7 °C; Rf
= 0.3 (ethyl acetate) [UV, KMnO4]; [α]24
589 –121.1 (c 0.5, CHCl3).
IR (NaCl): 3437, 3183, 2945, 2883, 2362, 2333, 1733, 1679, 1453, 1359, 1207, 1158,
754 cm–1.
1H NMR (360 MHz, CDCl3): δ = 7.35–7.27 (m, 3 H), 7.24–7.21 (m, 2 H), 6.90 (br s, 1 H), 5.61 (br s, 1 H),
3.92 (d, J = 13.1 Hz, 1 H), 3.84 (d, J = 13.1 Hz, 1 H), 3.77 (dd, J = 3.3 Hz, J = 10.9 Hz, 1 H), 3.74 (d, J = 7.7 Hz, 1 H), 3.69 (s, 3 H), 2.54 (dtd, J = 8.1 Hz, J = 10.9 Hz, J = 12.6 Hz, 1 H), 2.16–2.04 (m, 1 H), 2.00–1.85 (m, 2 H).
13C NMR (91 MHz, CDCl3): δ = 177.7, 173.5, 138.0, 128.7, 128.6, 127.6, 65.8, 62.9, 54.2, 51.4, 29.4, 28.6.
HRMS (ESI): m/z [M + Na]+ calcd for C14H18N2NaO3: 285.1209; found: 285.1202.
Methyl (2S,5S)-1-Benzyl-5-{[(tert-butoxycarbonyl)amino]methyl}pyrrolidine-2-carboxylate (16a)
Methyl (2S,5S)-1-Benzyl-5-{[(tert-butoxycarbonyl)amino]methyl}pyrrolidine-2-carboxylate (16a)
Borane dimethyl sulfide (2 M in dry tetrahydrofuran, 2.73 mL, 5.55 mmol) was added
dropwise to a solution of amide 15a (393 mg, 1.50 mmol) in dry tetrahydrofuran (7.6 mL) under an argon atmosphere and
the reaction mixture was stirred for 6 h at 50 °C. Subsequently, dry methanol (13
mL) was added dropwise and the reaction mixture was stirred for 18 h at 75 °C. The
solvent was removed under reduced pressure and the obtained residue was dissolved
in tetrahydrofuran (15 mL) and treated with potassium carbonate (212 mg, 2.00 mmol)
and di-tert-butyldicarbonate (437 mg, 2.00 mmol) for 8.5 h. Water (50 mL) was added and the resulting
mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic phases
were washed with a saturated aqueous solution of sodium chloride and dried over sodium
sulfate. The crude mixture was purified by column chromatography (silica gel, hexane/ethyl
acetate = 9:1 → 4:1 → ethyl acetate) to give the title compound 16a (392 g, 1.13 mmol, 75%) as a colorless oil.
Rf
= 0.5 (hexane/ethyl acetate = 4:1) [UV, KMnO4]; [α]24
589 –110.3 (c 0.5, CHCl3).
IR (NaCl): 3392, 2977, 2952, 1733, 1714, 1496, 1454, 1366, 1249, 1163, 743, 699 cm–1.
1H NMR (600 MHz, CDCl3): δ = 7.38–7.36 (m, 1 H), 7.32–7.28 (m, 2 H), 7.26–7.22 (m, 2 H), 4.79 (br s, 1 H),
3.91 (d, J = 13.5 Hz, 1 H), 3.72 (d, J = 13.5 Hz, 1 H), 3.66 (s, 3 H), 3.63 (d, J = 8.2 Hz, 1 H), 3.45–3.39 (m, 1 H), 3.34 (dd, J = 8.3 Hz, J = 12.2 Hz, 1 H), 3.05 (d, J = 13.1 Hz, 1 H), 2.16 (qd, J = 9.4 Hz, J = 12.6 Hz, 1 H), 2.05–1.98 (m, 1 H), 1.78 (dd, J = 9.8 Hz, J = 11.9 Hz, 1 H), 1.74-1.68 (m, 1 H), 1.45 (s, 9 H).
13C NMR (91 MHz, CDCl3): δ = 174.4, 156.4, 139.2, 128.5, 128.4, 127.1, 78.1, 63.3, 60.9, 52.9, 51.5, 42.1,
28.4, 28.1, 27.0.
HRMS (ESI): m/z [M + Na]+ calcd for C19H28NaN2O4: 371.1941; found: 371.1939.
(2S,5S)-1-{[(9H-Fluoren-9-yl)methoxy]carbonyl}-5-{[(tert-butoxycarbonyl)amino]methyl}pyrrolidine-2-carboxylic Acid (17a)
(2S,5S)-1-{[(9H-Fluoren-9-yl)methoxy]carbonyl}-5-{[(tert-butoxycarbonyl)amino]methyl}pyrrolidine-2-carboxylic Acid (17a)
A suspension of 16a (912 mg, 2.62 mmol) and palladium on carbon (10% w/w, 524 mg, 0.49 mmol) in dry ethyl
acetate (26 mL) was stirred at 45 °C for 2 h under a hydrogen atmosphere (1 bar).
The reaction course was monitored by TLC. After the reaction was complete, the reaction
mixture was filtered over Celite® and the filter cake was washed with ethyl acetate. The solvent was removed under
reduced pressure and (2S,5S)-methyl 5-{[(tert-butoxycarbonyl)amino]methyl}pyrrolidine-2-carboxylate (18) (quant.) was used without further purification. The obtained product (301 mg, 1.17 mmol)
was dissolved in methanol (12 mL), sodium hydroxide (2 M, 5.83 mL) was added, and
the reaction mixture was stirred for 2 h at room temperature. The reaction course
was monitored via TLC. Subsequently, the pH was adjusted to a value of 8 by addition
of hydrochloric acid (3 M) and a saturated aqueous solution of potassium carbonate.
The solvent was removed under reduced pressure and the residue was dissolved in tetrahydrofuran
(29 mL) and water (7.3 mL). Sodium bicarbonate (392 mg, 4.66 mmol) and 9-fluorenylmethoxycarbonyl
chloride (453 mg, 1.75 mmol) were added and the reaction mixture was stirred for 1
h at room temperature. The reaction course was monitored by TLC. After extraction
with ethyl acetate (2 × 50 mL), the combined organic phases were washed with a saturated
solution of sodium bicarbonate, dried over sodium sulfate and the remaining aqueous
phases were kept for further processing (see below). The solvent of the organic phase
was removed under reduced pressure leading to a residue containing (2S,5S)-1-{[(9H-fluoren-9-yl)methoxy]carbonyl}-5-{[(tert-butoxycarbonyl)amino]methyl}pyrrolidine-2-carboxylic [(9H-fluoren-9-yl)methyl-carbonic] anhydride. The residue containing the anhydride was
dissolved in tetrahydrofuran (29 mL) and water (7.3 mL) and the mixture was stirred
for 7 h at room temperature, while the reaction course was monitored by TLC. The solvent
was removed under reduced pressure, the residue was dissolved in water (30 mL) and
washed with methyl-tert-butyl ether (2 × 30 mL). The remaining aqueous phase was adjusted to a pH value of
2 with hydrochloric acid (3 M) and was extracted with ethyl acetate (4 × 40 mL). The
combined ethyl acetate phases were dried over sodium sulfate and the solvent was removed
under reduced pressure to give 17a (52%). Subsequently, the previously kept combined aqueous phases (see above) were
adjusted to a pH value of 2 with hydrochloric acid (3 M) and extracted with ethyl
acetate (4 × 70 mL). The combined organic phases of this extraction were dried over
sodium sulfate and the solvent was removed under reduced pressure to afford an additional
amount of 17a (27 mg, 0.06 mmol, 4%). The title compound 17a (304 mg, 94% w/w, 0.61 mmol, 52%, in total 56%) was obtained as a white solid.
Rf
= 0.3 (CH2Cl2/methanol, 19:1) [UV, ninhydrin].
IR (NaCl): 3354, 2976, 2360, 2341, 1699, 1520, 1450, 1415, 1365, 1340, 1247, 1166,
1128, 1089, 758, 741 cm–1.
1H NMR (600 MHz, CDCl3): δ (mixture of rotamers, 1:1) = 7.76 (dd, J = 3.7 Hz, J = 7.4 Hz, 1 H), 7.73 (d, J = 7.5 Hz, 1 H), 7.59–7.52 (m, 2 H), 7.44–7.27 (m, 4 H), 5.10 (t,
J
= 5.9 Hz, 0.5 H), 4.72–4.65 (m, 1 H), 4.50–4.43 (m, 1 H), 4.32–4.28 (m, 0.5 H), 4.26–4.21
(m, 1 H), 4.15–4.11 (m, 0.5 H), 4.08–4.01 (m, 1 H), 3.47–3.40 (m, 0.5 H), 3.33–3.26
(m, 0.5 H), 3.26–3.13 (m, 0.5 H), 2.84–2.77 (m, 0.5 H), 2.63–2.57 (m, 0.5 H), 2.35–1.76
(m, 4 H), 1.44 (s, 4.5 H), 1.42 (s, 4.5 H).
13C NMR (151 MHz, CDCl3): δ (mixture of rotamers, 1:1) = 175.6, 174.7, 156.4, 156.1, 155.4, 144.0, 143.8,
143.7, 143.6, 141.41, 141.36, 141.3, 127.84, 127.79, 127.7, 127.6, 127.4, 127.3, 127.1,
127.0, 124.9, 124.8, 124.6, 119.94, 119.91, 119.85, 79.5, 79.3, 67.3, 66.8, 59.6,
59.3, 58.9, 58.6, 47.3, 47.2, 43.7, 42.4, 28.8, 28.4, 27.4, 26.9, 26.7.
HRMS (ESI): m/z [M + H]+ calcd for C26H31N2O6: 467.2177; found: 467.2175.
Chiral HPLC: Method A: t
R = 30.49 min; Method B: t
R = 22.42 min.
Methyl (2S)-1-({[(9H-Fluoren-9-yl)methoxy]carbonyl}-l-valyl)-5-(aminomethyl)pyrrolidine-2-carboxylate (19)
Methyl (2S)-1-({[(9H-Fluoren-9-yl)methoxy]carbonyl}-l-valyl)-5-(aminomethyl)pyrrolidine-2-carboxylate (19)
A suspension of 16a (71.6 mg, 0.03 mmol) and palladium on carbon (10% w/w, 42 mg, 0.05 mmol) in dry ethyl
acetate (5 mL) was stirred at 45 °C under a hydrogen atmosphere (1 bar). The reaction
course was monitored by HPLC-MS. After the reaction was complete, the reaction mixture
was filtered over Celite® and the filter cake was washed with ethyl acetate. The solvent was removed under
reduced pressure and (2S,5S)-methyl 5-{[(tert-butoxycarbonyl)amino]methyl}pyrrolidine-2-carboxylate (18) (quant.) was used without further purification.
1H NMR (400 MHz, CDCl3): δ = 4.99 (s, 1 H), 3.83–3.78 (m, 1 H), 3.74 (s, 3 H), 3.43–4.39 (m, 1 H), 3.24–3.17
(m, 1 H), 3.04–2.96 (m, 1 H), 2.22–2.10 (m, 2 H), 1.90–1.81 (m, 2 H), 1.45 (s, 9 H).
For the synthesis of 19, compound 18 (20 mg, 0.08 mmol), Fmoc-l-Val-OH (102 mg, 0.3 mmol) and N,N-diisopropylethylamine (0.17 mL, 1.00 mmol) were dissolved in dry N,N-dimethylformamide (2 mL) before 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (0.4 mmol, 152 mg) was added and the
reaction mixture stirred overnight. The reaction course was monitored by HPLC. After
18 h, water was added and the reaction mixture was extracted with ethyl acetate (3 × 15
mL). The combined organic phases were washed with a saturated aqueous solution of
sodium chloride and dried over sodium sulfate. Subsequently, the solvent was removed
under reduced pressure. Prepurification by column chromatography on silica was conducted
using hexane/ethyl acetate = 1:1 → ethyl acetate/methanol = 9:1, during which cleavage
of the Boc group occurred on removal of the solvent. A second column chromatography
(silica, hexane/ethyl acetate = 4:1) provided 19 (23.1 mg, 0.05 mmol, 66%) as a colorless oil.
Rf
= 0.7 (hexane/ethyl acetate = 1:1) [UV, KMnO4].
1H NMR (400 MHz, CDCl3, acidic): δ = 7.79 (d, J = 7.5 Hz, 2 H), 7.63 (dd, J = 2.4 Hz, J = 7.5 Hz, 2 H), 7.43 (t, J = 7.5 Hz, 2 H), 7.38–7.32 (m, 3 H), 5.38–5.28 (m, 1 H), 4.55–4.40 (m, 2 H), 4.34
(dd, J = 4.9 Hz, J = 9.1 Hz, 1 H), 4.26 (t, J = 7.0 Hz, 1 H), 3.78 (s, 3 H), 2.24–2.15 (m, 1 H), 1.76-1.52 (m, 3 H), 1.45–1.43
(m, 1 H), 1.29–1.23 (m, 1 H), 1.15–1.10 (m, 3 H), 1.00 (d, J = 6.9 Hz, 3 H), 0.94 (d, J = 6.9 Hz, 3 H).
13C NMR (151 MHz, CDCl3, acidic): δ = 172.6 (Cq), 156.2 (Cq), 143.9 (Cq), 143.8 (2 × Cq), 141.3 (2 × Cq), 127.7 (2 × CH), 127.1 (2 × CH), 125.1 (2 × CH), 120.0 (2 × CH), 67.0 (CH2), 59.5 (CH2), 59.0 (CH), 52.2 (CH3), 47.2 (2 × CH), 38.2 (2 × CH2), 31.3 (CH), 31.2 (CH), 18.9 (CH3), 17.6 (CH3).
HRMS (ESI): m/z [M + H]+ calcd for C27H34N3O5: 480.2493; found: 480.2493.
