Synthesis and Spectroscopic Properties of Fluorinated Coumarin Lysine Derivatives

Article abstract of DOI:10.1021/acs.joc.7b03214

The site-selective incorporation of fluorescent amino acids into proteins has emerged as a valuable alternative to expressible protein reporters. For successful application, a robust and scalable, yet flexible, route to non-natural amino acids is required. This work describes an improved synthesis of coumarin-conjugated lysine derivatives where fluorinated variants are accessed. These analogues can be utilized at low pH and should find application probing biological processes that operate under acidic conditions.

Full text of DOI:10.1021/acs.joc.7b03214

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Synthesis and Spectroscopic Properties of
Fluorinated Coumarin Lysine Derivatives
Lakshmi Shukla, Lindon W. K. Moodie, Tomas Kindahl, and Christian Hedberg
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b03214 • Publication Date (Web): 29 Mar 2018
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Synthesis and Spectroscopic Properties of Fluorinated Coumarin
Lysine Derivatives
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Lakshmi Shukla, Lindon W. K. Moodie, Tomas Kindahl and Christian Hedberg*
Umeå University, Department of Chemistry, 90187 Umeå, Sweden
Eꢀmail:Christian.Hedberg@chem.umu.se
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TOC Graphics
ABSTRACT: The siteꢀselective incorporation of fluorescent amino acids into
proteins has emerged as a valuable alternative to expressible protein reporters. For
successful application, a robust and scalable yet flexible route to nonꢀnatural amino
acids is required. This work describes an improved synthesis of coumarinꢀconjugated
lysine derivatives where fluorinated variants are accessed. These analogues can be
utilized at low pH and should find application probing biological processes that
operate under acidic conditions.
Fluorescently labeled proteins are valuable tools for studying biological
processes. Green fluorescent protein is universally employed,1 but has limitations in
terms of its size and that it can only be placed at the termini of the studied protein,
which can perturb protein function. The siteꢀselective incorporation of nonꢀnatural
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fluorescent amino acids is a promising alternative to expressible protein tags due to
their significantly smaller size, higher photo efficiency and the option to introduce
them at internal positions of the protein of interest (Figure 1A).2ꢀ4 Unnatural amino
acids can be introduced in response to a tactically placed stop codon, typically the
least commonly used amber codon, TAG, by orthogonal tRNA/aminoacylꢀtRNA
synthetase pairs.5 Amber suppression studies have been successfully demonstrated in
prokaryotic,6 eukaryotic systems7 and in living organisms.8
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The coumarin structural motif is commonly incorporated into probes for
biological study due to its desirable photoꢀphysical properties: high quantum yields,
photostability (low bleaching) and reasonable Stokes shifts. Coumarins are also
amendable to functionalization, typically at the 3ꢀ or 4ꢀpositions.9 7ꢀ
Hydroxycoumarins exhibit pH dependent photophysical properties, dictated by the
pKa of the phenolic substituent (1, pKa = 7.7) and therefore operate more efficiently
under neutral to basic conditions. To investigate biological systems that operate under
reduced pH, for example the lysosomal and endosomal networks (pH ≈ 5), coumarins
that exhibit greater phenolic acidity are desirable. Aryl ring fluorination is an effective
strategy to achieve this, as is evident by the phenol pKa of diFMU (3) (pKa = 4.7), in
comparison to its parent derivative umbelliferone (2) (pKa = 7.8) (Figure 1B).10,11
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A)
pH 5.0
pH 7.8
pKa 7.7
O
O
OH
O
O
O
O
O
7
OH
5
6
7
8
O
O
Endosomal-
Lysosomal
localisation
HN
Recombinant
expression
Protein
of
interest
Protein
of
interest
9
NH3+Cl-
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CO2H
1
Weakly fluorescent at
endosomal pH (5) +99%
protonated at 7-OH
Fluorescent at
physiological pH (7.8)
B)
pKa 7.8
pKa 4.7
F
O
O
OH
F
O
O
OH
3
2
C)
pKa 7.7
pKa 6.2
pKa 4.7
pKa 4.0
F
F
F
O
O
OH
O
O
OH
F
O
O
OH
F
O
O
7
OH
F
O
O
O
O
O
O
O
O
HN
HN
HN
HN
NH3+Cl-
NH3+Cl-
NH3+Cl-
NH3+Cl-
CO2H
CO2H
CO2H
CO2H
1
4
5
6
Decreasing phenolic pKa
Increasing fluorescence at low pH
Figure 1. Dependence of fluorescence on phenolic coumarin pKa.
In 2014, Deiters’ group reported the synthesis of the novel coumarin functionalized
lysine 1. Using an engineered pyrrolysylꢀtRNA synthetase system, this amino acid
was incorporated into proteins in both bacteria and mammalian cells, where it was
used as a fluorescent probe for protein localization.12
Given the successful application of 1, and the effect of coumarin fluorination on
phenolic pKa, we envisaged that fluorinated variants 4ꢀ6 would find application in
elucidating bacterial translocation systems, as well as protein trafficking in the
endosome at lower pH (Figure 1C). However, for the required orthogonal
tRNA/aminoacylꢀtRNA synthetase mutagenesis studies, and further application in
expression systems that often exhibit low incorporation efficiency, ample amounts of
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amino acids 1 and 4-6 would be required. The current route to 1 is reported only on
the milligram scale, including a low yielding step (33%), which reduces the overall
synthetic efficiency of the route.12 Furthermore, it utilizes the methoxymethyl (MOM)
as a phenolic protecting group, which is installed with chloromethyl methyl ether, a
known carcinogen not acceptable in most laboratories. We hereby report a modified
approach that enables gram scale syntheses of coumarinꢀfunctionalized lysines 1 and
fluorinated analogues 4-6. Key facets of this route include the use of N,N’ꢀ
disuccinimidyl carbonate to unify the coumarin and lysine moieties, and a benzylꢀ
based protecting group strategy that allows easily manipulated synthetic intermediates
and global deprotection by hydrogenation to afford the desired compounds as their
hydrochloride salts.
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The synthesis commenced with the Pechmann condensation of resorcinols 7aꢀd with
diethyl 1,3ꢀacetonedicarboxylate to afford the requisite coumarin scaffolds 8a-d
(Scheme 1). Reaction of compounds 7a-c proceeded smoothly in methane sulfonic
acid resulting in coumarin esters 8a-c (76ꢀ91%). For 2,4,5ꢀtrifluoro resorcinol 7d, we
found that sulfuric acid was more suitable to affect the Pechmann reaction. As some
of the corresponding acid was detected along with the desired product, the crude
reaction mixture was treated with SOCl2/EtOH for reꢀesterification, which after
purification afforded coumarin ester 8d in 90% yield. During evaluation of the
previously reported route to 1,12 we found that the boraneꢀmediated reduction of a
coumarin acid that lacked protection of the Cꢀ7 phenolic functionality was
problematic due to overꢀreduction of the coumarin system, particularly on larger
scales.
Scheme 1. Synthesis of coumarin-lysines 1, 4-6.
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CO2Et
O
CO2Et
R3
CO2Et
O
R3
R1
R3
R1
MeSO3H,
rt to 70 oC, 4-24 h
R2
R2
R2
BnOH, DEAD
PPh3, THF
0 oC to rt,
5-15 min
Or
HO
OH
HO
O
BnO
O
O
1) H2SO4, 60 o
C
CO2Et
R1
2) SOCl2, EtOH,
0 oC to rt, 3 h
7a-d
8a: 91%
8b: 81%
8c: 76%
8d: 90%
9a: 79%
9b: 85%
9c: 91%
9d: 80%
a: R1 = R2 = R3 = H
b: R2 = F, R1 = R3 =H
c: R1, R2 = F, R3 = H
d: R1, R2, R3 = F
9
CO2H
OH
R3
R1
R3
CO(OSu)2
DMAP (0.15 equiv.)
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R2
BnO
R2
BH3 SMe2, THF
0 oC to rt
LiOH H2O
CH2Cl2, 0 oC to rt, 15 h
Dioxane:H2O
10 oC to rt, 17 h
O
O
BnO
O
O
R1
10a-d
11a: 63% (2 steps)
11b: 41% (2 steps)
11c: 39% (2 steps)
11d: 44% (2 steps)
O
NHCbz
O
NHCbz
CO2Bn
O
N
-Cl+H3N
13
CO2Bn
R3
O
O
N
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O
O
R3
3
3
H
O
R2
BnO
R2
BnO
NaHCO3, Dioxane:H2O
10 oC to rt, 0.5 h
O
O
R1
R1
14a: 78%
14b: 85%
14c: 79%
14d: 83%
12a: quant.
12b: quant.
12c: quant.
12d: quant.
F
F
O
O
OH
O
O
OH
F
O
O
OH
F
O
O
OH
F
F
H2, Pd/C
O
O
O
O
O
O
O
O
EtOH:H2O, HCl
0 oC-rt, 6 h
HN
HN
HN
HN
NH3+Cl-
CO2H
1 (94%)
NH3+Cl-
NH3+Cl-
NH3+Cl-
CO2H
4 (85%)
CO2H
CO2H
6 (88%)
5 (86%)
It was rationalized that this problem could be circumvented via 7ꢀOH benzylation of
the appropriate coumarin ester prior to reduction. However, standard conditions for
phenolic benzylation (BnBr, K2CO3, DMF) resulted in complex reaction mixtures,
which included compounds indicative of overꢀalkylation; monoꢀbenzylation at the αꢀ
carbon of the ethyl ester was observed (see Supporting Information). Introducing the
phenolic benzylꢀprotecting group under Mitsunobu condition solved this issue and the
corresponding benzylated coumarins 9aꢀd were isolated in excellent yields (79ꢀ91%),
Attempts were made to directly reduce ethyl ester 9a to the requisite alcohol 11a by
employing esterꢀselective reducing agents (e.g. Zn(BH4)2 or LiBH4). However, in both
cases the reactions were sluggish, while increasing temperature or reaction times
yielded complex reaction mixtures. Alternatively, ester hydrolysis using lithium
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hydroxide followed by acidification gave the corresponding coumarin acids, which on
reduction with BH3·Me2S, yielded coumarin alcohols 11a-d (63ꢀ44%) over two steps.
To access an intermediate suitable for linking the coumarin alcohol and lysine
moieties together, the previously reported route utilized pꢀnitrophenyl chloroformate
to form the corresponding mixed carbonate.12 We found that this reagent difficult to
control on larger scales, resulting in inconsistent yields. To remedy this, we decided
to employ N,N’ꢀdisuccinimidyl carbonate (SuO)2CO as a synthetic lynchpin between
the coumarin and lysine motifs. Treatment of coumarin alcohol 11c with (SuO)2CO
under standard conditions (DIPEA in MeCN) provided the corresponding elimination
product in close to quantitative yield (see SI). After careful investigation, it was found
that a subꢀstoichiometric amount (20 mol%) of 4ꢀdimethylaminopyridine (DMAP) in
CH2Cl2 successfully promoted the formation of mixed OSuꢀcarbonates 12aꢀd in high
yields and purity after aqueous workup. It should be noted that these intermediates are
reasonably stable and could be stored at ꢀ20°C without noticeable decomposition.
Unsymmetrical carbonates 12aꢀd were coupled to Cbzꢀprotected lysine benzyl ester
13 under mild conditions (dioxane/water, NaHCO3), providing the fully protected
coumarinꢀlysine derivatives 14aꢀd in high yields. With the fully protected amino acid,
the hydroxyꢀsuccinimide byproduct can be easily removed by aqueous workup.
Furthermore, these fully protected derivatives can be purified to absolute
homogeneity by column chromatography at this stage, prior to global deprotection.
Final deprotections of 14aꢀd were conducted using standard conditions (Pd/C,
EtOH/H2O, H2) with the inclusion of aq. HCl (1 eq.). After filtration, the reaction
mixtures were concentrated to dryness and triturated with diethyl ether to provide the
corresponding HClꢀsalts 1, 4ꢀ6 in excellent yields (Scheme 1).
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With the four coumarinꢀfunctionalized lysines in hand, we investigated their
spectroscopic properties from a range of pH values (1.0 – 10.8) to elucidate the effect
of fluorination. The phenolic pKa’s of compounds 1 and 4ꢀ6 were determined by
examining the effect of pH on absorbance at 360 nm, revealing a clear relationship
between increasing fluorine substitution and decreasing pKa (Table 1). The
compounds displayed favorable photophysical properties, including high quantum
yields, long lifetimes and wellꢀseparated emission peaks.
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Table 1. Spectroscopic data.a
Compound pKab emmax (nm)
Φf
τ (ns)
1
7.7
456
5.5
0.56
6.2
4.7
4.0
453
462
467
0.64
0.70
0.58
5.0
5.5
5.6
4
5
6
a For the deprotonated, fluorescent state. b Determined with absorption at 360 nm
Examining the normalized total fluorescence emission of 1 from pH 2.2 – 10.8
showed that the nonꢀfluorinated amino acid derivative only showed significant
fluorescence at pH values greater than 6.0 (Figure 2) when excited at 360 nm. In
comparison, the fluorinated analogues 4ꢀ6 demonstrated fluorescence at lower pH
ranges. For applications in biological settings that operate at reduced pH’s,
compounds 5 and 6 in particular would be suitable.
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1
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1.0
0.8
0.6
0.4
0.2
0.0
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0
2
4
6
8
10
12
pH
Figure 2. Normalized summed fluorescence emission of coumarin derivatives 1, 4-6
with sigmoidal curve fit.
The site selective incorporation of nonꢀnatural fluorescent amino acids into proteins is
a powerful tool for studying biological processes. For effective application of this
technology, robust and scalable synthetic methodologies are required to access the
requisite amino acids. Furthermore, these probes must operate efficiently in the
cellular locations of study. The current research describes the scalable synthesis of
four coumarinꢀfunctionalized lysines, including three fluorinated derivatives (4ꢀ6).
The latter compounds displayed desirable spectroscopic properties at low pH and will
find use for probing protein function in acidic environments.
EXPERIMENTAL SECTION
General
All reactions were performed in ovenꢀdried apparatus and under a nitrogen
atmosphere. Unless otherwise mentioned, all chemicals, reagents and solvents were
used as obtained from commercial suppliers. Melting points were obtained in open
capillary tubes and are uncorrected. NMR spectra were recorded on Bruker AVANCE
1
(at 400 MHz or 600 MHz) spectrometers. H NMR chemical shifts are reported in δ
values relative to tetramethylsilane and referenced to the residual solvent peak
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(CDCl3: δH = 7.26 ppm, DMSOꢀd6: δH = 2.50 ppm and 13C NMR chemical shift
values are relative to tetramethylsilane (δ = 0.00 ppm), CDCl3 (δ = 77.16 ppm),
DMSOꢀd6 (δ = 39.50 ppm). Infrared spectra were recorded as thin films. High
resolution mass spectra (HRMS) were recorded using electron spray ionization timeꢀ
ofꢀflight (ESIꢀtof) mode. Optical rotations were measured using a 1 mL cell with a 1
dm path length and are reported as [α]D20 (c = g/100 mL, solvent).
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Ethyl 2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetate (8a).13 To a 0 °C cooled and
well stirred mixture of resorcinol 7a (11.0 g, 100.0 mmol) and diethyl 1,3ꢀ
acetonedicarboxylate (22.2 g, 110.0 mmol) was added MeSO3H (100 mL) dropwise
over a period of 10 minutes, and the resultant reaction solution was stirred at 50 °C
for 4 h. It was thereafter allowed to cool to room temperature and poured onto
crushed ice. The obtained yellowish precipitate was filtered, washed with cold water
(3 × 50 mL) and pet. ether (3 × 30 mL). The obtained solid was dried under high
vacuum to get compound 8a as a yellowish solid (22.5 g, 91%). Mp 150–152 °C (lit14
1
155–157 °C); H NMR (600 MHz, DMSOꢀd6) δ 10.59 (s, 1H), 7.51 (d, J = 8.8 Hz,
1H), 6.80 (dd, J = 8.7, 2.4 Hz, 1H), 6.73 (d, J = 2.3 Hz, 1H), 6.23 (s, 1H), 4.11 (q, J =
7.1 Hz, 2H), 3.93 (s, 2H), 1.18 (t, J = 7.1 Hz, 3H); 13C NMR (150 MHz, DMSOꢀd6) δ
169.2, 161.3, 160.1, 155.0, 149.6, 126.7, 113.0, 112.1, 111.2, 102.3, 60.9, 36.9, 14.0.
Ethyl
2-(6-fluoro-7-hydroxy-2-oxo-2H-chromen-4-yl)acetate
(8b).
The
experimental procedure was similar to that for compound 8a, except that the resultant
reaction solution was heated at 70 °C for 24 h. After filtration, washing and drying,
1
compound 8b (4.30 g, 81%) was obtained as an offꢀwhite solid. Mp 160–163 °C; H
NMR (600 MHz, DMSOꢀd6) δ 11.16 (s, 1H), 7.55 (d, J = 11.5 Hz, 1H), 6.92 (d, J =
7.3 Hz, 1H), 6.32 (s, 1H), 4.12 (q, J = 7.1 Hz, 2H), 3.95 (s, 2H), 1.18 (t, J = 7.0 Hz,
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3H); 13C NMR (150 MHz, DMSOꢀd6) δ 169.0, 159.9, 150.6, 149.2 (d, J = 2.5 Hz),
149.1 (d, J = 14.4 Hz), 148.01 (d, J = 239.6 Hz), 113.2, 111.9 (d, J = 21.3 Hz), 110.7
(d, J = 7.6 Hz), 104.6 (d, J = 3.1 Hz), 60.8, 36.7, 14.0; 19F NMR (376 MHz, DMSOꢀ
d6) δ ꢀ139.15 (dd, J = 11.4, 7.3 Hz); IR (neat) 3176, 2985, 1739, 1681cmꢀ1; HRMS
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(ESI) calcd for C13H12FO5 (M + H)+, 267.0663; found, 267.0674.
+
Ethyl 2-(6,8-difluoro-7-hydroxy-2-oxo-2H-chromen-4-yl)acetate (8c). The
experimental procedure was similar to that for compound 8a, except that the resultant
reaction solution was stirred at room temperature overnight. After filtration, washing
and drying, compound 8c (4.00 g, 76%) was obtained as an offꢀwhite solid. Mp 143–
1
145 °C; H NMR (600 MHz, DMSOꢀd6) δ 11.58 (s, 1H), 7.47 (d, J = 11.3 Hz, 1H),
6.43 (s, 1H), 4.12 (q, J = 7.1 Hz, 2H), 3.97 (s, 2H), 1.19 (t, J = 7.1 Hz, 3H); 13C NMR
(150 MHz, DMSOꢀd6) δ 168.9, 158.6, 149.2, 148.4 (dd, J = 238.6. 5.2 Hz), 139.6 (d,
J = 9.1 Hz), 138.3 (dd, J = 244.6, 6.8 Hz), 137.7 (dd, J = 18.0, 12.8 Hz), 114.3, 110.2
(d, J = 9.2 Hz), 106.6 (dd, J = 21.9, 3.0 Hz), 61.0, 36.7, 13.9; 19F NMR (376 MHz,
DMSOꢀd6) δ ꢀ136.02 (d, J = 9.2 Hz), ꢀ153.55 (d, J = 9.3 Hz); IR (neat) 3476,1712,
+
1637 cmꢀ1; HRMS (ESI) calcd for C13H11F2O5 (M + H)+, 285.0569; found, 285.0567.
Ethyl 2-(5,6,8-trifluoro-7-hydroxy-2-oxo-2H-chromen-4-yl)acetate (8d). To a 0 °C
cooled and well stirred mixture of resorcinol 7d15 (1.64 g, 10.0 mmol) and diethyl
1,3ꢀacetonedicarboxylate (2.42 g, 12.0 mmol) was added conc. H2SO4 (10 mL) slowly
over a period of 10 minutes. The resultant reaction solution was stirred at 60 °C
overnight. It was thereafter allowed to cool to room temperature, poured onto crushed
ice, and extracted with ethyl acetate (3 × 50 mL). The combined organic phases were
washed with water (2 × 50 mL), brine (50 mL), dried over Na2SO4 and concentrated
under reduced pressure. The obtained residue was dissolved in EtOH (12.8 mL),
cooled to 0 °C, and SOCl2 (4.76 g, 40.0 mmol) was added dropwise. The resultant
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reaction solution was stirred at room temperature for 3 h. The volatiles were removed
under reduced pressure and the residue was extracted with EtOAc (3 × 40 mL),
washed with water (2 × 30 mL), brine, dried over Na2SO4 and concentrated under
reduced pressure. The obtained residue was triturated with pet. ether to give
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compound 8d (2.70 g, 90%) as an offꢀwhite solid. Mp 201–203 °C; H NMR (600
MHz, DMSOꢀd6) δ 12.27 (br s, 1H), 6.47 (s, 1H), 4.13 (q, J = 7.1 Hz, 2H), 3.98 (d, J
= 4.3 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H); 13C NMR (150 MHz, DMSOꢀd6) δ 169.0,
157.8, 146.6 (m), 143.5 (ddd, J = 249.3, 12.2, 3.5 Hz), 138.7 (td, J = 14.0, 4.3 Hz),
138.4 (ddd, J = 10, 8, 2 Hz), 137.9 (dd, J = 256.3, 5.3 Hz), 136.3 (ddd, J = 241.2, 5, 3
19
Hz), 115.7, 100.9 (d, J = 13.4 Hz), 60.8, 39.3, 14.0; F NMR (376 MHz, DMSOꢀd6)
δ ꢀ144.68 (ddd, J = 21.9, 9.5, 4.6 Hz), ꢀ158.62 (dd, J = 9.8, 5.4 Hz), ꢀ162.09 (dd, J =
21.9, 5.3 Hz); IR (neat) 3226, 2986, 1728, 1647 cmꢀ1; HRMS (ESI) calcd for
+
C13H10F3O5 (M + H)+, 303.0475; found, 303.0476.
Ethyl 2-(7-(benzyloxy)-2-oxo-2H-chromen-4-yl)acetate (9a).13 To a cooled 0 °C
solution of compound 8a (6.20 g, 25.0 mmol) in anhydrous THF (250 mL) were
added benzyl alcohol (4.59 g, 42.5 mmol), diethyl azodicarboxylate (DEAD, 40% in
toluene, 16.03 mL, 35.0 mmol) and PPh3 (9.83 g, 37.5 mmol). The resultant reaction
solution was stirred at 0 °C for 15 min. After this, volatiles were removed under
reduced pressure and the residue was purified by silica gel column chromatography
(5–50% EtOAc in pet. ether) to get compound 9a (6.65 g, 79%) as a white solid. Mp
1
138–140 °C; H NMR (400 MHz, CDCl3) δ 7.48 (d, J = 8.8 Hz, 1H), 7.44–7.31 (m,
5H), 6.92 (dd, J = 8.8, 2.5 Hz, 1H), 6.88 (d, J = 2.5 Hz, 1H), 6.21 (s, 1H), 5.11 (s,
13
2H), 4.18 (q, J = 7.1 Hz, 2H), 3.70 (s, 2H), 1.24 (t, J = 7.1 Hz, 3H); C NMR (100
MHz, CDCl3) δ 168.8, 161.9, 160.9, 155.5, 148.2, 135.78, 128.8, 128.4, 127.6, 125.7,
113.9, 113.2, 112.7, 102.2, 70.54, 61.79, 38.30, 14.13.
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Ethyl 2-(7-(benzyloxy)-6-fluoro-2-oxo-2H-chromen-4-yl)acetate (9b). The
experimental procedure was similar to that for compound 9a. After column
purification, compound 9b (1.52 g, 85%) was obtained as a white solid. Mp 126–128
°C; 1H NMR (600 MHz, CDCl3) δ 7.46– 7.26 (m, 6H), 6.94 (d, J = 7.1 Hz, 1H), 6.27
(s, 1H), 5.18 (s, 2H), 4.19 (q, J = 7.2 Hz, 2H), 3.67 (s, 2H), 1.26 (t, J = 7.1 Hz, 3H);
13C NMR (150 MHz, CDCl3) δ 168.5, 160.4, 151.0 (d, J = 1.9 Hz), 150.2 (d, J = 12.7
Hz), 149.32 (d, J = 245.6 Hz), 147.7 (d, J = 2.7 Hz), 135.1, 128.9, 128.6, 127.5,
115.0, 111.8 (d, J = 7.4 Hz), 110.8 (d, J = 21.5 Hz), 103.2 (d, J = 2.0 Hz), 71.4, 61.9,
38.3, 14.1; 19F NMR (376 MHz, CDCl3) δ ꢀ136.75 (dd, J = 11.2, 7.1 Hz); IR (neat)
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1728, 1626, 1563 cmꢀ1; HRMS (ESI) calcd for C20H18FO5 (M + H)+, 357.1133;
+
found, 357.1136.
Ethyl 2-(7-(benzyloxy)-6,8-difluoro-2-oxo-2H-chromen-4-yl)acetate (9c). The
experimental procedure was similar to that for compound 9a. After column
purification, compound 9c (0.34 g, 91%) was obtained as a white solid. Mp 92–94 °C;
1H NMR (600 MHz, CDCl3) δ 7.46–7.41 (m, 2H), 7.40–7.30 (m, 3H), 7.09 (dd, J =
10.9, 2.2 Hz, 1H), 6.36 (s, 1H), 5.33 (s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 3.67 (s, 2H),
1.26 (t, J = 7.1 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 168.2, 158.8, 151.8 (dd, J =
246.5, 4.4 Hz), 147.2 (t, J = 2.7 Hz), 143.6 (dd, J = 253.7, 6.0 Hz), 140.1 (dd, J =
10.5, 2.7 Hz), 138.4 (dd, J = 15.6, 10.8 Hz), 135.7, 129.0, 128.8, 128.4, 117.1, 114.1
(d, J = 9.2 Hz), 105.9 (dd, J = 22.6, 3.7 Hz), 76.3 (t, J = 3.8 Hz), 62.2, 38.4, 14.2; 19F
NMR (376 MHz, CDCl3) δ ꢀ130.42 (d, J = 5.3 Hz), ꢀ145.07 (d, J = 5.2 Hz).; IR (neat)
+
1734, 1629, 1511 cmꢀ1; HRMS (ESI) calcd for C20H17F2O5 (M + H)+, 375.1039;
found, 375.1039.
Ethyl 2-(7-(benzyloxy)-5,6,8-trifluoro-2-oxo-2H-chromen-4-yl)acetate (9d). To
anhydrous THF (10 mL) at 0 °C were added diethyl azodicarboxylate (DEAD, 40% in
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toluene, 0.5 mL, 1.1 mmol), PPh3 (335 mg, 1.3 mmol), compound 8d (302 mg, 1.0
mmol), and benzyl alcohol (0.13 mL, 1.2 mmol). The resultant reaction solution was
stirred at 0 °C for 5 min, after which volatiles were removed under vacuum. The
residue was purified by silica gel column chromatography (5–50% EtOAc in pet.
ether) to get compound 9d (314 mg, 80%) as a white solid. Mp 141–143 °C; 1H NMR
(600 MHz, CDCl3 ) δ 7.44 (d, J = 7.3 Hz, 2H), 7.42–7.34 (m, 3H), 6.24 (s, 1H), 5.37
(s, 2H), 4.21 (q, J = 7.1 Hz, 2H), 3.80 (d, J = 4.3 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H);
13C NMR (150 MHz, CDCl3) δ 168.6, 157.9, 145.6 (q, J = 2.2 Hz), 144.0 (ddd, J =
252.2, 13.7, 4.2 Hz), 141.1 (ddd, J = 247.6, 16.0, 4.5), 139.9 (dt, J = 250.2, 4.0 Hz),
139.0 (d, J = 3.6 Hz), 138.9 (dd, J = 4.6, 3.1 Hz), 135.4, 129.2, 128.9, 128.4, 118.0,
105.0 (d, J = 12.9 Hz), 76.5, 61.9, 40.5 (d, J = 8.8 Hz), 14.2; 19F NMR (376 MHz,
CDCl3) δ ꢀ143.38 (ddt, J = 20.2, 10.6, 4.5 Hz), ꢀ151.45 (d, J = 10.7 Hz), ꢀ156.29 (d, J
= 20.7 Hz); IR (neat) 1719, 1644, 1503 cmꢀ1; HRMS (ESI) calcd for C20H16F3O5+ (M
+ H)+, 393.0944; found, 393.0945.
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7-(Benzyloxy)-4-(2-hydroxyethyl)-2H-chromen-2-one (11a).13 To a cooled solution
of 9a (7.30 g, 21.6 mmol) in dioxane:H2O (130 mL, 5:1) was added LiOH:H2O (2.90
g, 69.1 mmol) and the resultant reaction solution was stirred at room temperature for
17 h. All volatiles were removed under reduced pressure, and the residue was
dissolved in water. The resultant solution was cooled to 0 °C and then acidified with
HCl (6 M, 15.0 mL) to pH 2–3. The aqueous layer was extracted with EtOAc (3 ×
100 ml), and the combined organic layers were washed with water (2 × 100 ml) and
brine (100 ml), and dried over Na2SO4. The organic phase was concentrated under
reduced pressure to give carboxylic acid 10a (6.60 g) as a yellow solid. The crude
product was used asꢀis for the following reaction procedure.
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The obtained acid was dissolved in anhydrous THF (198 mL) and cooled to 0 °C.
BH3ꢁSMe2 (1.78 g, 23.4 mmol) was added dropwise, and resultant reaction solution
was stirred at room temperature for 16 h. The reaction mixture was cooled to 0 °C and
quenched with MeOH (60 mL). All volatiles were removed under vacuum, and the
residue was purified by silica gel column chromatography (35–90% EtOAc in pet.
ether) to give alcohol 11a (4.00 g, 63% over two steps) as a yellowish solid. Mp 130–
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132 °C; H NMR (600 MHz, CDCl3) δ 7.55 (d, J = 8.9 Hz, 1H), 7.43–7.31 (m, 5H),
6.93 (dd, J = 8.8, 2.5 Hz, 1H), 6.87 (d, J = 2.5 Hz, 1H), 6.20 (s, 1H), 5.10 (s, 2H),
3.97 (t, J = 6.4 Hz, 2H), 2.98 (t, J = 6.5 Hz, 2H); 13C NMR (150 MHz, CDCl3) δ
161.8, 161.5, 155.6, 153.5, 135.9, 128.9, 128.5, 127.6, 125.6, 113.2, 113.2, 112.1,
102.2, 70.6, 60.9, 34.9.
7-(Benzyloxy)-6-fluoro-4-(2-hydroxyethyl)-2H-chromen-2-one
(11b).
The
experimental procedure was similar to that for compound 11a. After column
purification, compound 11b (650 mg, 41% over two steps) was obtained as a
1
yellowish solid. Mp 112–114 °C; H NMR (600 MHz, CDCl3) δ 7.49–7.30 (m, 6H),
6.92 (d, J = 7.1 Hz, 1H), 6.26 (s, 1H), 5.18 (s, 2H), 3.98 (t, J = 6.3 Hz, 2H), 2.93 (td, J
= 6.3, 1.0 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 161.1, 152.9 (d, J = 2.7 Hz), 151.0
(d, J = 1.9 Hz), 150.1 (d, J = 12.8 Hz), 149.44 (d, J = 245.4 Hz), 135.2, 129.0, 128.7,
127.6, 113.2, 112.4 (d, J = 7.2 Hz), 110.7 (d, J = 21.3 Hz), 103.3 (d, J = 2.0 Hz), 71.5,
60.7, 34.9; 19F NMR (376 MHz, CDCl3) δ ꢀ136.96 (dd, J = 11.3, 7.2 Hz); IR (neat)
+
3387, 1760, 1722, 1564 cmꢀ1; HRMS (ESI) calcd for C18H16FO4 (M + H)+, 315.1027;
found, 315.1034.
7-(Benzyloxy)-6,8-difluoro-4-(2-hydroxyethyl)-2H-chromen-2-one (11c). The
experimental procedure was similar to that for compound 11a. After column
purification, compound 11c (1.90 g, 39% over two steps) was obtained as a yellowish
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solid. Mp 109–111 °C; H NMR (600 MHz, CDCl3) δ 7.48–7.41 (m, 2H), 7.40–7.31
(m, 3H), 7.15 (dd, J = 11.1, 2.3 Hz, 1H), 6.35 (s, 1H), 5.33 (s, 2H), 3.99 (t, J = 6.2 Hz,
2H), 2.92 (td, J = 6.3, 1.1 Hz, 2H), 1.75 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ
159.4, 152.6 (t, J = 2.7 Hz), 151.8 (dd, J = 244, 4.4 Hz), 143.5 (dd, J = 253.4, 6.0 Hz),
140.0 (dd, J = 10.3, 2.5 Hz), 138.1 (dd, J = 15.7, 10.7 Hz), 135.7, 129.0, 128.7, 128.4,
115.0, 114.6 (d, J = 8.9 Hz), 105.8 (dd, J = 22.4, 3.7 Hz), 76.3 (t, J = 3.8 Hz), 60.4,
34.7; 19F NMR (376 MHz, CDCl3) δ ꢀ130.78 (dd, J = 11.1, 5.4 Hz), ꢀ145.25 (d, J =
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5.2 Hz).; IR (neat) 3423, 1724, 1630, 1567 cmꢀ1; HRMS (ESI) calcd for C18H15F2O4
+
(M + H)+, 333.0933; found, 333.0940.
7-(Benzyloxy)-5,6,8-trifluoro-4-(2-hydroxyethyl)-2H-chromen-2-one (11d). The
experimental procedure was similar to that for compound 11a. After column
purification, compound 11d (430 mg, 44% over two steps) was obtained as a
1
yellowish solid. Mp 111–113 °C; H NMR (600 MHz, CDCl3 ) δ 7.43 (d, J = 6.6 Hz,
2H), 7.41–7.32 (m, 3H), 6.31 (s, 1H), 5.37 (s, 2H), 3.97 (t, J = 6.0 Hz, 2H), 3.11–3.07
(m, 2H), 1.94 (br 2, 1H); 13C NMR (151 MHz, CDCl3) δ 158.2, 151.0, 144.2 (ddd, J =
253.3, 13.6, 3.9 Hz), 141.3 (ddd, J = 247.3, 16.0, 4.6 Hz), 139.9 (dt, J = 250.3, 3.8
Hz), 139.1 (dd, J = 17.9, 2.9 Hz), 138.7 (td, J = 12.1, 3.4 Hz), 135.3, 129.1, 129.0,
128.4, 116.3, 105.1 (d, J = 12.5 Hz), 76.4, 60.5 (d, J = 4.0 Hz), 37.8 (d, J = 9.0 Hz);
19F NMR (376 MHz, CDCl3) δ ꢀ142.57 – ꢀ142.84 (m), ꢀ151.45 (d, J = 10.8 Hz), ꢀ
156.27 (d, J = 20.5 Hz); IR (neat) 3424, 1745, 1642 cmꢀ1; HRMS (ESI) calcd for
C18H14F3O4 (M + H)+, 351.0839; found, 351.0830.
+
2-(7-(Benzyloxy)-2-oxo-2H-chromen-4-yl)ethyl
2,5-dioxopyrrolidin-1-yl
carbonate (12a). To a cooled 0 °C suspension of N,N′ꢀdisuccinimidyl carbonate (3.63
g, 14.2 mmol) in anhydrous CH2Cl2 (135 ml) were added compound 11a (4.0 g, 13.5
mmol) and 4ꢀdimethylaminopyridine (0.25 g, 2.03 mmol). The resultant reaction
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mixture was allowed to warm to room temperature, and stirred overnight. The mixture
was partitioned between CH2Cl2 (100 ml) and water (100 ml), separated, and the
aqueous layer was extracted with CH2Cl2 (2 × 70 mL). The combined organic layers
were washed with 5% citric acid (2 × 70 ml), saturated NaHCO3 (2 × 100 ml), water
(2 × 100 ml) and brine (100 ml), and dried over Na2SO4. The solution was
concentrated under reduced pressure to give carbonate 12a (5.8 g, quantitative) as an
off white solid. Mp 70–72 °C; 1H NMR (400 MHz, CDCl3) δ 7.52 (d, J = 8.8 Hz, 1H),
7.46–7.33 (m, 5H), 6.97 (dd, J = 8.8, 2.5 Hz, 1H), 6.91 (d, J = 2.6 Hz, 1H), 6.19 (s,
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1H), 5.13 (s, 2H), 4.60 (t, J = 6.9 Hz, 2H), 3.18 (t, J = 6.9 Hz, 2H), 2.83 (s, 4H); C
NMR (100 MHz, CDCl3) δ 168.6, 162.1, 160.7, 155.7, 151.6, 150.3, 135.8, 128.9,
128.5, 127.6, 125.2, 113.5, 112.9, 112.5, 102.5, 70.6, 68.4, 30.8, 25.6; IR (neat) 1812,
+
1788, 1737, 1611 cmꢀ1; HRMS (ESI) calcd for C23H20NO8 (M + H)+, 438.1183;
found, 438.1185.
2-(7-(Benzyloxy)-6-fluoro-2-oxo-2H-chromen-4-yl)ethyl-2,5-dioxopyrrolidin-1-yl
carbonate (12b). The experimental procedure was similar to that for compound 12a.
The compound 12b (455 mg, quantitative) was obtained as an offꢀwhite solid. Mp
1
172–175 °C; H NMR (400 MHz, CDCl3 ) δ 7.45 (d, J = 7.6 Hz, 2H), 7.41 (t, J = 7.5
Hz, 2H), 7.36 (t, J = 7.2 Hz, 1H), 7.32 (d, J = 11.0 Hz, 1H), 6.97 (d, J = 6.9 Hz, 1H),
6.26 (s, 1H), 5.21 (s, 2H), 4.60 (t, J = 6.8 Hz, 2H), 3.14 (t, J = 6.8 Hz, 2H), 2.84 (s,
4H); 13C NMR (151 MHz, CDCl3) δ 168.5, 160.4, 151.6, 151.2, 150.4 (d, J = 4.6 Hz),
149.7 (d, J = 2.7 Hz), 149.6 (d, J = 241.6 Hz), 135.2, 129.0, 128.8, 127.6, 114.1,
111.6 (d, J = 7.1 Hz), 110.3 (d, J = 21.4 Hz), 103.6, 71.6, 68.0, 30.9, 25.6; 19F NMR
(376 MHz, CDCl3) δ ꢀ136.36 (dd, J = 11.0, 7.1 Hz); IR (neat) 1812, 1788, 1739, 1628
cmꢀ1; HRMS (ESI) calcd for C23H19FNO8 (M + H)+, 456.1089; found, 456.1071.
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2-(7-(Benzyloxy)-6,8-difluoro-2-oxo-2H-chromen-4-yl)ethyl-2,5-dioxopyrrolidin-
1-yl carbonate (12c). The experimental procedure was similar to that for compound
12a. The compound 12c (1.85 g, quantitative) was obtained as an off white solid. Mp
85–88 °C; 1H NMR (400 MHz, CDCl3 ) δ 7.46–7.42 (m, 2H), 7.38–7.33 (m, 3H), 7.13
(dd, J = 10.9, 2.0 Hz, 1H), 6.34 (s, 1H), 5.33 (s, 2H), 4.59 (t, J = 6.7 Hz, 2H), 3.12 (t,
J = 6.7 Hz, 2H), 2.82 (s, 4H); 13C NMR (151 MHz, CDCl3) δ 168.6, 158.6, 151.9 (dd,
J = 241.6, 4.3 Hz), 151.5, 149.4 (t, J = 2.7 Hz), 143.7 (dd, J = 254.0, 5.9 Hz), 140.2
(dd, J = 10.2, 2.5 Hz), 138.4 (dd, J = 15.6, 10.7 Hz), 135.7, 128.9, 128.8, 128.4,
115.9, 113.8 (d, J = 8.8 Hz), 105.3 (dd, J = 22.6, 3.4 Hz), 76.3 (t, J = 3.8 Hz), 67.8,
30.8, 25.5; 19F NMR (376 MHz, CDCl3) δ ꢀ130.14 (d, J = 5.7 Hz), ꢀ144.66 (d, J = 5.9
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Hz); IR (neat) 1813, 1789, 1741, 1570 cmꢀ1; HRMS (ESI) calcd for C23H18F2NO8 (M
+
+ H)+, 474.0995; found, 474.0983.
2-(7-(Benzyloxy)-5,6,8-trifluoro-2-oxo-2H-chromen-4-yl)ethyl-2,5-
dioxopyrrolidin-1-yl carbonate (12d). The experimental procedure was similar to
that for compound 12a. The compound 12d (490 mg, quantitative) was obtained as an
1
off white solid. Mp 162–164 °C; H NMR (400 MHz, CDCl3 ) δ 7.44 (d, J = 7.1 Hz,
2H), 7.41–7.36 (m, 3H), 6.31 (s, 1H), 5.39 (s, 2H), 4.62 (t, J = 6.0 Hz, 2H), 3.20–3.36
(m, 2H), 2.82 (s, 4H); 13C NMR (151 MHz, CDCl3) δ 168.5, 157.5, 151.6, 147.8,
143.9 (ddd, J = 252.0, 13.5, 4.1 Hz), 141.2 (ddd, J = 241.6, 15.7, 4.4 Hz), 140.0 (dt, J
= 250.7, 3.9 Hz), 139.3–138.9 (m, presumably overlap of 2 C and one of the triplets
from the dt at 140.0 ppm), 135.3, 129.2, 129.0, 128.4, 117.7, 104.4 (d, J = 12.6 Hz),
76.5 (t, J = 3.8 Hz), 68.0, 34.1 (d, J = 8.7 Hz), 25.6; 19F NMR (376 MHz, CDCl3) δ ꢀ
143.8 (dd, J = 20.7, 10.7 Hz), ꢀ150.9 (d, J = 10.7 Hz), ꢀ156.0 (d, J = 20.6 Hz); IR
(neat) 1789, 1742, 1643 cmꢀ1; HRMS (ESI) calcd for C23H17F3NO8 (M + H)+,
+
492.0901; found, 492.0871.
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(S)-Benzyl-6-((2-(7-(benzyloxy)-2-oxo-2H-chromen-4-yl)ethoxy)carbonylamino)-
2-(benzyloxycarbonylamino)hexanoate (14a). To a cooled solution of lysine
hydrochloride 13 (5.41 g, 13.3 mmol) in dioxane:H2O (8:2, 266 mL) were added
NaHCO3 (2.2 g, 26.6 mmol) and carbonate 12a (5.8 g, 13.3 mmol). The resultant
reaction mixture was slowly allowed to warm to room temperature and stirred for 0.5
h. After the completion of the reaction, EtOAc (150 mL) and water (80 mL) were
added, and the organic layer was separated. The aqueous layer was extracted with
EtOAc (3 × 70 mL), and the combined organic layers were washed with water (2 × 70
mL) and brine (100 mL), dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by silica gel column chromatography (35–
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90% EtOAc in pet. ether) to give carbamate 14a (7.1 g, 78%) as a yellowish sticky
1
solid; [α]20 = ꢀ2.6 (c = 1.0, CHCl3); H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 8.6
D
Hz, 1H), 7.46–7.27 (m, 15H), 6.93 (dd, J = 8.8, 2.5 Hz, 1H), 6.87 (d, J = 2.5 Hz, 1H),
6.14 (s, 1H), 5.53 (d, J = 8.2 Hz, 1H), 5.25–5.02 (m, 6H), 4.88–4.80 (m, 1H), 4.46–
4.35 (m, 1H), 4.30 (t, J = 6.7 Hz, 2H), 3.15–3.03 (m, 2H), 2.98 (t, J = 6.6 Hz, 2H),
1.91–1.76 (m, 1H), 1.75–1.60 (m, 1H), 1.55–1.38 (m, 2H), 1.37–1.24 (m, 2H); 13C
NMR (150 MHz, CDCl3) δ 172.3, 161.8, 161.2, 156.3, 156.1, 155.5, 152.3, 136.3,
135.8, 135.4, 128.8, 128.7, 128.57, 128.56, 128.5, 128.4, 128.2, 128.1, 127.6, 125.5,
113.2, 112.9, 112.3, 102.3, 70.5, 67.2, 67.1, 62.3, 53.8, 40.5, 32.1, 31.7, 29.3, 22.3; IR
+
(neat) 3332, 3033, 1720, 1611 cmꢀ1; HRMS (ESI) calcd for C40H41N2O9 (M + H)+,
693.2807; found, 693.2810.
(S)-Benzyl-6-((2-(7-(benzyloxy)-6-fluoro-2-oxo-2H-chromen-4-
yl)ethoxy)carbonylamino)-2-(benzyloxycarbonylamino)hexanoate (14b). The
experimental procedure was similar to that for compound 14a. After column
purification, compound 14b (0.6 g, 85%) was obtained as a yellowish solid. Mp 78–
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80 °C; [α]20D = ꢀ3.0 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3 ) δ 7.44 (d, J = 7.6
Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.37–7.30 (m, 12H), 6.92 (d, J = 7.0 Hz, 1H), 6.19
(s, 1H), 5.48 (d, J = 8.3 Hz, 1H), 5.21–5.05 (m, 6H), 4.76 (t, J = 6.0 Hz, 1H), 4.44–
4.36 (m, 1H), 4.30 (t, J = 6.6 Hz, 2H), 3.17–2.99 (m, 2H), 2.93 (t, J = 6.6 Hz, 2H),
1.89–1.78 (m, 1H), 1.74–1.62 (m, 1H), 1.53–1.41 (m, 2H), 1.37–1.26 (m, 2H); 13C
NMR (150 MHz, CDCl3) δ 172.3, 160.8, 156.14 (d, J = 5.0 Hz), 151.9, 151.0 (d, J =
1.5 Hz), 150.1 (d, J = 12.7 Hz), 149.5 (d, J = 245.5 Hz), 136.3, 135.4, 135.2, 129.0,
128.74, 128.70, 128.62, 128.60, 128.4, 128.3, 128.2, 127.6, 113.4, 112.1 (d, J = 7.3
Hz), 110.6 (d, J = 21.3 Hz), 103.4 (d, J = 1.8 Hz), 71.5, 67.19 (d, J = 15.0 Hz), 62.1,
53.8, 40.6, 32.2, 31.7, 29.3, 22.3; 19F NMR (376 MHz, CDCl3) δ ꢀ136.7; IR (neat)
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3332, 2949, 1719 cmꢀ1; HRMS (ESI) calcd for C40H40FN2O9 (M + H)+, 711.2712;
+
found, 711.2721.
(S)-Benzyl-6-((2-(7-(benzyloxy)-6,8-difluoro-2-oxo-2H-chromen-4-
yl)ethoxy)carbonylamino)-2-(benzyloxycarbonylamino)hexanoate (14c). The
experimental procedure was similar to that for compound 14a. After column
purification, compound 14c (1.9 g, 79%) was obtained as a yellowish sticky solid.
1
[α]20 = ꢀ2.8 (c = 1.0, CHCl3); H NMR (400 MHz, CDCl3 ) δ 7.44 (d, J = 6.9 Hz,
D
2H), 7.41–7.27 (m, 13H), 7.15 (d, J = 10.8 Hz, 1H), 6.27 (s, 1H), 5.46 (d, J = 7.9 Hz,
1H), 5.32 (s, 2H), 5.22–5.12 (m, 2H), 5.08 (s, 2H), 4.76 (br s, 1H), 4.47– 4.35 (m,
1H), 4.29 (s, 2H), 3.11 (s, 2H), 3.02–2.81 (m, 2H), 1.91–1.76 (m, 1H), 1.75–1.60 (m,
1H), 1.55–1.40 (m, 2H), 1.40–1.24 (m, 2H); 13C NMR (150 MHz, CDCl3) δ 172.3,
159.0, 156.1, 152.7 (d, J = 4.2 Hz), 151.3, 151.1 (d, J = 4.2 Hz), 144.4 (d, J = 5.8 Hz),
142.8 (d, J = 6.0 Hz), 140.1 (d, J = 10.1 Hz), 138.2 (dd, J = 15.8, 10.9 Hz), 136.3,
135.7, 135.4, 128.9, 128.77, 128.75, 128.6, 128.45, 128.41, 128.3, 128.2, 115.3, 114.3
(d, J = 8.9 Hz), 105.6 (dd, J = 24.2, 2.6 Hz), 76.2 (t, J = 3.7 Hz), 67.3, 67.1, 61.8,
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53.8, 40.6, 32.2, 31.7, 29.3, 22.2; 19F NMR (376 MHz, CDCl3) δ ꢀ130.46 (d, J = 5.4
Hz), ꢀ145.18 (d, J = 5.3 Hz); IR (neat) 3332, 2949, 1724 cmꢀ1; HRMS (ESI) calcd for
C40H39F2N2O9 (M + H)+, 729.2618; found, 729.2610.
+
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(S)-Benzyl-6-((2-(7-(benzyloxy)-5,6,8-trifluoro-2-oxo-2H-chromen-4-
yl)ethoxy)carbonylamino)-2-(benzyloxycarbonylamino)hexanoate (14d). The
experimental procedure was similar to that for compound 14a. After column
purification, compound 14d (756 mg, 83%) was obtained as a yellowish sticky solid.
1
[α]20 = ꢀ2.6 (c = 1.0, CHCl3); H NMR (400 MHz, CDCl3) δ 7.47–7.27 (m, 15H),
D
6.20 (s, 1H), 5.52 (d, J = 8.0 Hz, 1H), 5.37 (s, 2H), 5.16 (q, J = 12.3 Hz, 2H), 5.08 (s,
2H), 4.81 (br. s, 1H), 4.52–4.26 (m, 3H), 3.20–3.01 (s, 4H), 1.92–1.77 (m, 1H), 1.73–
1.62 (m, 1H), 1.52–1.21 (m, 4H); 13C NMR (151 MHz, CDCl3) δ 172.3, 158.0, 156.1,
150.2, 144.0 (ddd, J = 253.3, 13.1, 3.8 Hz), 141.2 (ddd, J = 247.8, 15.9, 4.2 Hz),
139.8 (dt, J = 250.0, 3.3 Hz), 139.1ꢀ138.5 (m, presumably overlap of 2 C and one of
the triplets from the dt at 139.8 ppm), 136.3, 135.4, 135.3, 129.1, 128.7 (d, J = 19.2
Hz), 128.54, 128.52, 128.4, 128.3, 128.2, 128.1, 116.3, 104.8 (d, J = 12.6 Hz), 98.9,
76.4 (t, J = 3.4 Hz), 67.2, 67.0, 66.1 (d = 19.9 Hz), 61.7 (d, J = 3.4 Hz), 61.0, 53.8,
40.5, 34.6 (d, J = 8.9 Hz), 32.0, 29.3, 22.3; 19F NMR (376 MHz, CDCl3) δ ꢀ142.78, ꢀ
142.81, ꢀ142.83, ꢀ142.86, ꢀ151.41, ꢀ151.44, ꢀ156.13, ꢀ156.18; IR (neat) 3359, 2943,
+
1723, 1700 cmꢀ1; HRMS (ESI) calcd for C40H38F3N2O9 (M + H)+, 747.2524; found,
747.2533.
(S)-2-amino-6-(((2-(7-hydroxy-2-oxo-2H-chromen-4-
yl)ethoxy)carbonyl)amino)hexanoic acid hydrochloride (1).12 To a cooled
suspension of carbamate 14a (7.10 g, 10.3 mmol) in EtOH:H2O (9:1, 103 mL) was
added conc. HCl (0.96 mL, 1 eq.) followed by Pd/C (10% w/w, 0.71 g). The reaction
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mixture was evacuated and backꢀfilled with H2, and was stirred at room temperature
under an H2 atmosphere (1 atm). After completion, the reaction solution was filtered
through a small pad of celite and washed with EtOH:H2O (1:1, 2 × 50 mL). The
filtrate was concentrated under reduced pressure, and the residue was triturated with
Et2O. The obtained solid was dissolved in H2O (60 mL) and lyophilized to give
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compound 1 (4.00 g, 94%) as a white solid. Mp 185–188 °C; [α]D = +10 (c = 1.0,
1
DMSO); H NMR (400 MHz, DMSOꢀd6) δ 8.32 (br s, 3H), 7.68 (d, J = 8.7 Hz, 1H),
7.17 (br s, 1H), 6.84 (d, J = 8.7 Hz, 1H), 6.76 (s, 1H), 6.11 (s, 1H), 4.31–4.21 (m,
2H), 3.84–3.70 (m, 1H), 3.08–3.00 (m, 2H), 2.99–2.90 (m, 2H), 1.84–1.70 (m, 2H),
1.45–1.20 (m, 4H); 13C NMR (100 MHz, DMSOꢀd6) δ 171.0, 161.4, 160.2, 156.0,
155.1, 153.5, 126.4, 113.1, 111.1, 110.4, 102.5, 61.6, 51.8, 31.0, 29.6, 28.8, 21.6; IR
+
(neat) 3371, 2933, 1688, 1602 cmꢀ1; HRMS (ESI) calcd for C18H23N2O7 (M + H)+,
379.1500; found, 379.1524.
(S)-2-amino-6-(((2-(6-fluoro-7-hydroxy-2-oxo-2H-chromen-4-
yl)ethoxy)carbonyl)amino)hexanoic acid hydrochloride (4). The experimental
procedure was similar to that for compound 1. Compound 4 (772 mg, 85%) was
1
obtained as a white solid. Mp 200–203 °C; [α]20D = +9.8 (c = 1.0, DMSO); H NMR
(600 MHz, DMSOꢀd6) δ 8.36 (s, 3H), 7.69 (d, J = 11.7 Hz, 1H), 7.18 (t, J = 5.3 Hz,
1H), 7.01 (d, J = 7.4 Hz, 1H), 6.19 (s, 1H), 4.23 (t, J = 6.2 Hz, 2H), 3.81 (t, J = 5.6
Hz, 1H), 3.01 (t, J = 6.2 Hz, 2H), 2.98–2.89 (m, 2H), 1.86–1.66 (m, 2H), 1.42–1.23
13
(m, 4H); C NMR (150 MHz, DMSOꢀd6) δ 171.0, 160.0, 156.0, 153.1, 150.5, 149.2
(d, J = 14.3 Hz), 148.2 (d, J = 239.8 Hz), 111.6, 111.5 (d, J = 21.2 Hz), 110.6 (d, J =
7.5 Hz), 104.7 (d, J = 2.9 Hz), 61.5, 51.9, 31.0, 29.6, 28.8, 21.6; 19F NMR (376 MHz,
DMSOꢀd6) δ ꢀ139.20 (dd, J = 11.2, 7.9 Hz); IR (neat) 2943, 1691, 1628 cmꢀ1; HRMS
+
(ESI) calcd for C18H22FN2O7 (M + H)+, 397.1406; found, 397.1411.
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(S)-2-amino-6-(((2-(6,8-difluoro-7-hydroxy-2-oxo-2H-chromen-4-
4
5
6
yl)ethoxy)carbonyl)amino)hexanoic acid hydrochloride (5). The experimental
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procedure was similar to that for compound 1. Compound 5 was obtained as a white
1
solid (505 mg, 86%). Mp 198–200°C; [α]20 = +10 (c = 1.0, DMSO); H NMR (600
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D
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MHz, DMSOꢀd6) δ 8.37 (s, 3H), 7.60 (d, J = 11.3 Hz, 1H), 7.18 (t, J = 5.1 Hz, 1H),
6.29 (s, 1H), 4.23 (t, J = 5.9 Hz, 2H), 3.79 (br. s, 1H), 3.03 (t, J = 5.8 Hz, 2H), 2.97–
2.88 (m, 2H), 1.83–1.67 (m, 2H), ), 1.43–1.20 (m, 4H); 13C NMR (150 MHz, DMSOꢀ
d6) δ 171.1, 158.7, 156.0, 153.2, 148.7 (dd, J = 239.5, 4.8 Hz), 139.6 (d, J = 9.0 Hz),
139.5 (dd, J = 240.0, 6.0 Hz), 138.0 (t, J = 15.9 Hz), 112.5, 110.0 (d, J = 9.0 Hz),
106.2 (d, J = 21 Hz), 61.4, 51.9, 31.1, 29.7, 28.8, 21.6; 19F NMR (565 MHz, DMSOꢀ
d6) δ ꢀ135.65, ꢀ135.66, ꢀ135.68, ꢀ153.61, ꢀ153.62; IR (neat) 2944, 1693, 1632 cmꢀ1;
+
HRMS (ESI) calcd for C18H21F2N2O7 (M + H)+, 415.1311; found, 415.1338.
(S)-2-amino-6-(((2-(5,6,8-trifluoro-7-hydroxy-2-oxo-2H-chromen-4-
yl)ethoxy)carbonyl)amino)hexanoic acid hydrochloride (6). The experimental
procedure was similar to that for compound 1. Compound 6 was obtained as a white
1
solid (210 mg, 88%). Mp 183–186 °C; [α]20 = +4 (c = 0.5, DMSO); H NMR (600
D
MHz, DMSOꢀd6) δ 8.30 (s, 3H), 7.16 (t, J = 5.2 Hz, 1H), 6.15 (s, 1H), 4.25 (t, J = 5.7
Hz, 2H), 3.79 (t, J = 5.7 Hz, 1H), 3.12–3.04 (s, 2H), 2.97–2.88 (m, 2H), 1.85–1.60
(m, 2H), 1.44–1.15 (m, 4H); 13C NMR (150 MHz, DMSOꢀd6) δ 171.0, 158.2, 155.8,
151.4, 144.1 (dd, J = 250.0, 13.3 Hz), 142.1, 138.9 (ddd, J = 241.6, 8.7, 4.8 Hz),
138.7 (t, J = 18.1 Hz), 137.10 (d, J = 237.6 Hz), 112.1, 98.7 (d, J = 9.9 Hz), 61.5 (d, J
19
= 3.9 Hz), 52.0, 34.0 (d, J = 8.7 Hz), 29.7, 28.8, 21.6; F NMR (376 MHz, DMSOꢀ
d6) δ ꢀ144.85, ꢀ160.30, ꢀ162.58 (d, J = 22.3 Hz); IR (neat) 2941, 1756, 1692, 1644 cmꢀ
1; HRMS (ESI) calcd for C18H20F3N2O7 (M + H)+, 433.1217; found, 433.1215.
+
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(S)-6-(Benzyloxy)-5-(benzyloxycarbonylamino)-6-oxohexan-1-amine:hydrogen
chloride salt (13).16,17 ZꢀLys(Boc)ꢀOH (0.38 g, 1 mmol) and cesium carbonate (0.39
g, 1.2mmol) were suspended in DMF (10 mL), stirred for 30 min, and cooled to 0 °C.
BnBr was added dropwise while stirring at 0 °C. After stirring vigorously for 2 hours
at room temperature, the reaction mixture was poured into water, extracted with
EtOAc, washed with 5% LiCl, water and brine, and dried over Na2SO4. The solvent
was evaporated under reduced pressure and triturated with Et2O to give ZꢀLys(Boc)ꢀ
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OBn as a white solid (0.46 g, 98%). Mp 92–94 °C; H NMR (400 MHz, CDCl3 ) δ
7.46–7.26 (m, 10H), 5.40 (d, J = 8.2 Hz, 1H), 5.25–4.98 (m, 4H), 4.63–4.26 (m, 2H),
3.05 (q, J = 7.0, 6.4 Hz, 2H), 1.90–1.78 (m, 1H), 1.75–1.61 (m, 1H), 1.51–1.22 (m,
13H); 13C NMR (100 MHz, CDCl3) δ 172.4, 156.14, 156.07, 136.3, 135.4, 128.8,
128.6, 128.4, 128.30, 128.26, 67.3, 67.1, 53.9, 40.1, 32.3, 29.7, 28.5, 22.4.
To a cooled solution of the obtained compound (0.41 g, 0.88 mmol) in CH2Cl2 (3.5
mL) was added HCl in dioxane (4 M, 3.0 mL, 12.3 mmol) dropwise. The resulting
mixture was stirred at room temperature for 4 h and was then concentrated under
reduced pressure. After trituration with Et2O, the title compound was obtained as
white sticky solid (0.33 g, 92%). 1H NMR (400 MHz, DMSOꢀd6) δ 7.98 (s, 3H), 7.80
(d, J = 7.7 Hz, 1H), 7.46 – 7.16 (m, 10H), 5.18 – 4.94 (m, 4H), 4.10–4.02 (m, 1H),
2.71 (q, J = 7.1 Hz, 2H), 1.76 – 1.45 (m, 4H), 1.42 – 1.29 (m, 2H); 13C NMR (100
MHz, DMSOꢀd6) δ 172.2, 156.2, 136.8, 135.9, 128.4, 128.34, 128.0, 127.85, 127.78,
127.75, 66.3, 65.9, 65.5, 53.9, 38.3, 30.0, 26.4, 22.4.
Acknowledgments
We thank Knut and Alice Wallenberg Foundation (Sweden, grant KAW 2013.0187)
for generous support. L. S. and L. W. K. M. thank the Kempe Foundation (Sweden)
for postdoctoral fellowships. We kindly thank Prof. Lennart Johansson (Umeå
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University, Sweden) and the Swedish Defense Research Agency (Dr. Magnus
Engqvist, FOI, Umeå, Sweden) for access to instrumentation for spectroscopic
analysis.
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Supporting Information Available: Copies of H NMR, 13C NMR, 19F NMR and
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related discussion. This material is available free of charge via the Internet at
http://www.pubs.acs.org.
Author Information
Corresponding author
*Eꢀmail: Christian.Hedberg@chem.umu.se
ORCID
Lakshmi Shukla: 0000ꢀ0001ꢀ8063ꢀ4756
Lindon W. K. Moodie: 0000ꢀ0002ꢀ9500ꢀ4535
Tomas Kindahl: 0000ꢀ0002ꢀ6548ꢀ6158
Christian Hedberg: 0000ꢀ0002ꢀ0832ꢀ8276
Notes: The authors declare no competing financial interests.
References
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(2)
(3)
(4)
(5)
(6)
Tsien, R. Y. Ann. Rev. Biochem. 1998, 67, 509.
Krueger, A. T.; Imperiali, B. ChemBioChem 2013, 14, 788.
Harkiss, A. H.; Sutherland, A. Org. Biomol. Chem. 2016, 14, 8911.
Sinkeldam, R. W.; Greco, N. J.; Tor, Y. Chem. Rev. 2010, 110, 2579.
Lang, K.; Chin, J. W. Chem. Rev. 2014, 114, 4764.
Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Science 2001, 292, 498.
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The Journal of Organic Chemistry
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