Design and synthesis of 3,3′-biscoumarin-based c-Met inhibitors

Article abstract of DOI:10.1039/c4ob00364k

A library of biscoumarin-based c-Met inhibitors was synthesized, based on optimization of 3,3′-biscoumarin hit 3, which was identified as a non-ATP competitive inhibitor of c-Met from a diverse library of coumarin derivatives. Among these compounds, 38 and 40 not only showed potent enzyme activities with IC₅₀ values of 107 nM and 30 nM, respectively, but also inhibited c-Met phosphorylation in BaF3/TPR-Met and EBC-1 cells. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00364k

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Biomolecular Chemistry
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Design and synthesis of 3,3’-biscoumarin-based
c-Met inhibitors†
Cite this: Org. Biomol. Chem., 2014,
12, 3721
Jimin Xu,‡a Jing Ai,‡b Sheng Liu,a Xia Peng,b Linqian Yu,a Meiyu Geng*b and
Fajun Nan*a
A library of biscoumarin-based c-Met inhibitors was synthesized, based on optimization of 3,3’-biscou-
marin hit 3, which was identified as a non-ATP competitive inhibitor of c-Met from a diverse library of
coumarin derivatives. Among these compounds, 38 and 40 not only showed potent enzyme activities
with IC50 values of 107 nM and 30 nM, respectively, but also inhibited c-Met phosphorylation in
BaF3/TPR-Met and EBC-1 cells.
Received 17th February 2014,
Accepted 3rd April 2014
DOI: 10.1039/c4ob00364k
www.rsc.org/obc
Introduction
c-Met is a receptor tyrosine kinase that is normally activated by its
natural ligand hepatocyte growth factor/scatter factor (HGF/SF).1
The HGF/c-Met axis plays an important role in normal
embryonic development and organ regeneration. However,
aberrant c-Met activation has been frequently found in many
human solid tumours and hematologic malignancies. Overac-
tivation of c-Met is known to initiate tumorigenesis and
promote metastasis, and also cause therapeutic resistance.2–5
Importantly, both c-Met and HGF elevation have been associ-
ated with poor clinical outcome or metastatic progression in
many major human cancers.6–9 As a result, c-Met is considered
to be a potential target for cancer treatment.
A variety of approaches have previously been used to target
Met signaling. These include HGF antagonists,10–12 anti-HGF
humanized antibodies,13 and MET extracellular domain mono-
clonal antibodies.14,15 Additionally, a large number of small-
molecule kinase inhibitors targeting c-Met are now in clinical
trials; most of them target the ATP binding site in an ATP-com-
petitive manner.3,16–18 Here, we report our efforts toward the
development of 3,3′-biscoumarin analogues as novel, potent
and non-ATP-competitive kinase inhibitors.
Fig. 1 Daphnetin derivatives.
including PKA (IC50 = 9.33 μM) and PKC (IC50 = 25.01 μM).19
During our initial efforts to synthesize a diverse library of cou-
marin derivatives, we found that the simple dimeric analogue
3 displayed potent c-Met inhibitory activity with an IC50 of
151 nM. Daphnetin 1 and 4-hydroxyl daphnetin 2, in contrast,
showed weak inhibitory activity (IC50 = 100 μM). Compound 3,
which has a novel structure type compared to other reported
c-Met inhibitors, is a dimer of 2 through a one-carbon linker
(Fig. 1). Its acetoxy derivative has been reported as a tool to
study protein transacetylase,20 and similar coumarin dimers
with different linkers have been reported as Hsp90 inhibitors
by Blagg et al.21,22 The c-Met inhibitory activities of these com-
pounds, however, have not previously been reported. As most
kinase inhibitors to date are ATP competitive, we examined
whether compound
3 functions in a similar manner.
PF2341066, a typical ATP-competitive inhibitor, was used as a
reference control.23 In contrast to PF2341066, the IC50 values
of 3 remained unchanged with increasing ATP concentration.
This suggests that 3 is an ATP non-competitive inhibitor of
c-Met (Fig. 2). These initial results encouraged us to pursue a
medicinal chemistry program to further optimize 3 as a novel
c-Met inhibitor.
Daphnetin 1, a derivative of coumarin, is a protein kinase
inhibitor which inhibits tyrosine-specific protein kinase, EGFR
(IC50 = 7.67 μM), and serine/threonine-specific protein kinases,
aChinese National Center for Drug Screening, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of Science, 189 Guoshoujing
Road, Zhangjiang Hi-Tech Park, Shanghai 201203, Republic of China.
E-mail: fjnan@simm.ac.cn
bDivision of Antitumor Pharmacology, Shanghai Institute of Materia Medica, Chinese
Results and discussion
Academy of Science, 555 Zuchongzhi Road, Shanghai 201203, Republic of China.
E-mail: mygeng@simm.ac.cn
†Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ob00364k
‡These two authors contributed equally to this work.
To explore the SAR of 3, simple modifications were made to its
structure. These changes, as shown in Scheme 1, yielded
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treated with formaldehyde in ethanol to provide the corres-
ponding dimers (3, 11a, 5, 11b, 8, respectively). 11a was con-
verted into compound 4 via methylation with diazomethane
and subsequent debenzylation using H2/Pd(OH)2. Deprotec-
tion of 11b under acidic conditions afforded compound 7.
The Pechmann reaction of pyrogallol 12 with compound 13 in
70% sulfuric acid provided compound 6 directly.
Compounds 4 and 6 showed potent inhibitory activities,
with IC50 of 112 nM and 63 nM respectively. Modifying the bis-
(7,8-dihydroxyl) moiety (R3 and R4, Table 1) with hydrogen or
methoxy groups led to a complete loss of potency (8 and 5).
Moving the phenolic hydroxyl groups from the 8,8′-position to
the 5,5′-position (R2), as in compound 7, also led to a large
loss of activity (IC50 = 3.5 μM). These results indicate that
retaining the bis-(7,8-dihydroxy) moiety of 3 is important for
maintaining its inhibitory activity, and that methoxy and
methyl groups are well-tolerated at the C-4,4′ position (R1).
Next, we explored c-Met’s inhibitory activity as it relates
to the linker between the two coumarin moieties (Table 2).
Compounds 14–18 were prepared as outlined in Scheme 2.
Coumarin acids 20 and 25 were formed by reaction of com-
pounds 19 and 24, respectively, with isopropylidene malonate
and piperidinium acetate in ethanol. Aminocoumarin 21 was
similarly accessed from 19 via a nitrocoumarin intermediate,
which was converted into aminocoumarin 21 by hydrogen-
ation. With intermediates 20, 21, and 25 in hand, the desired
compounds were readily synthesized by a series of conden-
sations and deprotections. To this end, condensation of amino-
coumarin 21 with acid 20 using EDCI in 30% pyridine/CH2Cl2
afforded compound 14 upon acidic deprotection.24 Com-
pounds 15 and 16 were accessed under the same coupling
Fig. 2 Compound 3 is an ATP non-competitive inhibitor of Met kinase
activity. Inhibition assays with recombinant c-Met protein and different
concentrations of 3 (A) or ATP-competitive PF2341066 (B) were per-
formed in the presence of various concentrations of ATP.
Scheme 1 Synthesis of biscoumarin compounds 3–8. Reagents and
conditions: (a) CO(OEt)2, NaH, toluene, 80 °C; (b) HCl–AcOEt; (c) para-
formaldehyde, Et3N, EtOH; (d) i. CH2N2, Et2O; ii. Pd(OH)2, H2, THF;
(e) 70% H2SO4.
Table 2 c-Met enzymatic activity of compounds 14–18
compounds 3–8 (Table 1). To this end, condensation of differ-
ently substituted starting materials 9a–e with diethyl carbonate
in the presence of sodium hydride formed coumarin monomers
10a–e. Subsequent deprotection of 10b under acidic conditions
gave monomer 2. The monomers (2, 10a, 10c–e) were then
Compound
X
IC50 a (nM)
14
620.8 70.9
Table 1 c-Met enzymatic activity of compounds 3–8
15
16
168.7 18.6
134.1 6.3
Compound
R1
R2
R3
R4
IC50 a (nM)
17
18
1370.9 208.5
122.0 20.3
3
4
5
6
7
8
OH
OCH3
OH
Me
OH
H
H
H
H
OH
H
OH
OH
OCH3
OH
OH
H
OH
OH
OCH3
OH
H
150.9 5.8
112.2 23.6
0%@10 μM
62.5 6.9
3545.7 159.7
0%@10 μM
OH
H
a IC50s were calculated by the logit method from the results of at least
two independent tests with eight concentrations each and expressed as
means SD.
a IC50s were calculated by the logit method from the results of at least
two independent tests with eight concentrations each and expressed as
means SD.
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Table 3 c-Met enzymatic activity of compounds 30–50
Compound
R1
R2
IC50 a (nM)
30
31
32
33
34
35
36
37
38
39
40
41
H
H
H
H
H
Et
n-Pr
n-Bu
i-Bu
H
3620.7 444.1
62.5 5.4
40.8 3.7
72.2 1.2
70.9 13.6
21.9 1.2
36.6 2.6
168.8 7.0
107.0 1.3
48.3 13.1
30.2 0.7
38.8 6.3
Me
n-Pr
i-Pr
n-Bu
H
H
H
H
n-Pen, H
n-Hex, H
Me
Me
42
43
115.4 8.4
H
H
H
129.0 14.0
44
45
21.7 0.7
24.5 0.8
Scheme 2 Synthesis of biscoumarin compounds 14–18 with modified
linkers. Reagents and conditions: (a) isopropylidene malonate, piperidi-
nium acetate, EtOH, 60 °C; (b) 1-Boc-piperazine, EDCI, DMAP, DCM;
(c) HCl–AcOEt; (d) 20, EDCI, DMAP, DCM; (e) Pd/C, H2, AcOEt; (f) ethyl
nitroacetate, piperidine, benzene, Dean–Stark trap, reflux; (g) EDCI,
30% pyridine, DCM; (h) piperidine, CH3CN; (i) HCOOH; ( j) TFA, DCM.
46
47
H
H
121.8 10.2
14.9 4.2
48
49
50
H
H
H
90.6 1.7
62.5 5.4
4805.8 1300.9
conditions, by condensation of 21 with 0.5 equiv. of the corres-
ponding di-acids 22 and 23, respectively, followed by the
removal of the MOM groups. Condensation of 25 with 1-Boc-
piperazine was followed by Boc deprotection under acidic con-
ditions to provide intermediate 26. Subsequent condensation
of 26 with coumarin acid 20 afforded 27, which was converted
to compound 17 via hydrogenolytic cleavage and acid depro-
tection. Fmoc-Leu-OH 28 was also condensed with 21 to give
an intermediate which was transformed to 29 via piperidine
deprotection. The coupling reaction between 29 and 20 was
followed by acid deprotection to give compound 18.
Biological testing revealed that compounds with cyclo-
hexane-1,2-dicarboxamide linkers (15, 16) had potent inhibi-
tory activities, with IC50 of 169 nM and 134 nM respectively.
Compounds with piperazine-1,4-diyl (17) and amide linkers
(14) displayed reduced potency, with IC50 of 1.40 μM and
0.62 μM respectively. Use of L-leucine as a linker (18) retained
potency against c-Met (IC50 = 122 nM). These results indicate
that the length of the linker can be adjusted and that substi-
tution on the linker has a great impact on inhibitory activity.
Considering the inhibitory activity of 18, we further
explored the effect of the substitution on the linker moiety
using various α-amino acids. Compounds 30–50 were syn-
thesized by a method analogous to that used to access 18,
starting from different Fmoc-protected amino acids
(Scheme 2). Their biological activities are shown in Table 3.
a IC50s were calculated by the logit method from the results of at least
two independent tests with eight concentrations each and expressed as
means SD.
Compounds 30–45 were evaluated to determine the effect of
size and chirality of linker substitution on c-Met inhibitory
potency. Use of an unsubstituted glycine linker (30, R1 = R2 = H)
resulted in weak inhibitory activity (IC50 = 3.6 μM). Alkyl
substitution significantly increased potency compared to 30;
linkers with (S)-methyl, dimethyl, (R)-ethyl, and (S)-isopropyl
substitution (31, 41, 35, 33) displayed potent c-Met inhibition
with IC50 of 63 nM, 39 nM, 22 nM and 72 nM, respectively.
Compounds 42–45 with cycloalkyl or benzyl substitution also
showed potent inhibition (IC50 = 22–130 nM). While the size of
the alkyl group is of significant importance to the enzymatic
inhibition potency, its configuration proved unimportant.
Compound 38 ((R)-isobutyl, IC50 = 107 nM) was as potent as its
epimer 18 ((S)-isobutyl, IC50 = 122 nM); (R)- and (S)-n-propyl
substituted compounds 32 and 36 also showed similar inhibi-
tory potencies (IC50 = 41 nM and 37 nM, respectively), while 37
((R)-n-butyl, IC50 = 169 nM) was slightly less potent than 34
((S)-n-butyl, IC50 = 71 nM). Racemic compounds 39 and 40
with n-Pen and n-Hex substituents displayed potent inhibitory
activities with IC50 of 48 nM and 30 nM respectively.
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Analogues 46–50 investigated the effect of heteroatom intro-
Table 4 c-Met enzymatic activity of compounds 51–55
duction on the side chain. Compound 46, which bears a
thioether, retained potency (IC50 = 122 nM) in comparison
with 34. The ester analogue (47, IC50 = 15 nM) offered potent
inhibitory activity, presenting 8-fold higher potency than the
corresponding acid (48, IC50 = 119 nM). For nitrogen-bearing
substituents, the amide analogue (49) retained inhibitory
potency (IC50 = 63 nM) relative to acid 48. The amine analogue
(50), however, showed 65-fold lower potency (IC50 = 4.8 μM)
than 34.
Using D-leucine as a linker, the effect of modifying the
hydroxyl groups on the two coumarin rings was explored. Com-
pounds 51–55 were synthesized according to the procedures
outlined in Scheme 3. Known compounds 56a–c were trans-
formed into coumarin acids 59a–c and aminocoumarins
21 and 57a by condensation. Coupling of Fmoc-D-Leu-OH with
57a and 21, followed by piperidine deprotection, provided
58a–b respectively. Condensation of 58a–b with 59a–c followed
by deprotection afforded compounds 51–55.
Compound
R1
R2
R3
IC50 a (nM)
51
52
53
54
55
OH
OH
OH
H
OCH3
H
OH
H
OCH3
OH
H
OH
H
51.8%@10 μM
1370.9 208.5
927.9 98.7
0%@10 μM
H
OH
0%@10 μM
a IC50s were calculated by the logit method from the results of at least
two independent tests with eight concentrations each and expressed as
means SD.
As shown in Table 4, methylation of two hydroxyl groups on
one coumarin ring (51) led to a significant loss of potency.
Removal of a hydroxyl group (52, 53) decreased the inhibitory
effects on c-Met, with IC50 of 1.37 μM and 0.93 μM, respect-
ively. Upon removal of one hydroxyl group on each coumarin
ring (54, 55), no inhibition of the enzyme expressing the c-Met
receptor was observed. These results are consistent with our
initial modification results (Table 1), which showed that the
existence of four hydroxyl groups is important to retain good
inhibitory activity.
Subsequently, compounds with different structures were
selected to evaluate their effect on c-Met phosphorylation in
BaF3/TPR-Met and EBC-1 NSCLC cell lines. BaF3/TPR-Met
cells stably express a constitutively active, ligand-independent,
oncogenic form of c-Met derived from chromosomal rearrange-
ment, whereas EBC-1 NSCLC cells harbor amplified MET
genes. As shown in Fig. 3, at the concentration of 10 μM,
38 and 40 markedly inhibited c-Met phosphorylation in both cell
lines; 42 and 48 only effectively inhibited c-Met phosphoryl-
ation in EBC-1 NSCLC cells. Other compounds (3, 35 and 52)
Fig. 3 The effect of selected compounds on c-Met phosphorylation in
EBC-1 and BaF3/TPR-Met cells.
failed to inhibit c-Met phosphorylation in either cell line. The
poor cell potencies of these compounds can be attributed to
their poor permeability, as they have many hydrophilic groups.
The relatively good cell potencies of 38 and 40, in contrast, can
be ascribed to improved liposolubility due to their large alkyl
substituents (isobutyl and n-Hex, respectively).
Conclusions
In summary, we have developed a series of 3,3′-biscoumarin-
based, non-ATP competitive c-Met inhibitors initiated from
3,3′-methylenebis(4,7,8-trihydroxy-coumarin). Among these
compounds, 38 and 40 showed potent enzyme activities with
IC50 of 107 nM and 30 nM respectively. Significantly, they
inhibit c-Met phosphorylation in BaF3/TPR-Met and EBC-1
NSCLC cell lines. These compounds represent a novel struc-
tural type for non-ATP competitive c-Met inhibitors, and are
Scheme 3 Synthesis of compounds 51–55 with D-leucine linker.
Reagents and conditions: (a) (i) ethyl nitroacetate, piperidine, benzene,
Dean–Stark trap, reflux; (ii) Pd/C, H2, AcOEt; (b) (i) Fmoc-D-Leu-OH,
EDCI, 30% pyridine, DCM; (ii) piperidine, CH3CN. (c) isopropylidene
malonate, piperidinium acetate, EtOH, 60 °C; (d) (i) EDCI, DMAP, DCM;
(ii) HCl–AcOEt.
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worth developing further thorough investigation of the SAR.
1-(2-Hydroxy-3,4-bis(methoxymethoxy)phenyl)ethanone (9b).
Such efforts are currently underway, and will be reported in Compound 9b was prepared utilizing the same synthetic route
due course.
as compound 9d starting from 1-(2,3,4-trihydroxyphenyl)etha-
none. Yellow oil (2.3 g, 60%). 1H NMR (300 MHz, CDCl3)
δ 12.63 (s, 1H), 7.48 (d, J = 9.0 Hz, 1H), 6.71 (d, J = 8.7 Hz, 1H),
5.27 (s, 2H), 5.19 (s, 2H), 3.63 (s, 3H), 3.50 (s, 3H), 2.57 (s, 3H).
1-(2-Hydroxy-3,4-dimethoxyphenyl)ethanone
pound 9c was prepared utilizing the same synthetic route as
Experimental section
Chemistry
(9c).26 Com-
Starting materials, reagents, and solvents were purchased from compound 56c starting from 1-(2,3,4-trihydroxyphenyl)etha-
commercial suppliers and used without further purification, none. 1H NMR (300 MHz, CDCl3) δ 12.97 (s, 1H), 7.49 (d,
unless otherwise stated. Anhydrous THF, benzene, diethyl J = 9.0 Hz, 1H), 6.50 (d, J = 9.0 Hz, 1H), 3.98 (s, 3H), 3.92 (s, 3H),
ether and CH2Cl2 were obtained by distillation over sodium 2.57 (s, 3H).
wire or CaH2. All non-aqueous reactions were run under an
1-(2-Hydroxy-4,6-bis(methoxymethoxy)phenyl)ethanone
argon atmosphere with exclusion of moisture from reagents, (9d).27 To a mixture of 1-(2,4,6-trihydroxyphenyl)ethanone
and all reaction vessels were oven-dried. The progress of reac- (2.15 g, 12.8 mmol) and DIPEA in 50 mL DCM at 0 °C was
tions was monitored by TLC on SiO2. Spots were visualized by added MOMCl (2.14 mL, 28.13 mmol) dropwise. After stirring
UV or by dipping into KMnO4 solution followed by heating. for 2 h at 0 °C, the mixture was diluted with 100 mL DCM and
SiO2 for flash chromatography was of 230−400 mesh particle washed with 10% aqueous citric acid (2 × 30 mL) and brine.
size. Petroleum ether refers to the fraction with boiling range The organic layer was dried with Na2SO4 and concentrated
60–90 °C. 1H NMR spectra were recorded on a Varian Mercury- under reduced pressure. The residue was purified through
Vx 300M Fourier transform spectrometer at a frequency of column chromatography (eluent, PE–EtOAc = 10 : 1) to afford
1
1
300 MHz, and 13C NMR spectra at 75 MHz. H chemical shifts 9d as a beige solid (2.1 g, 64.1%). H NMR (300 MHz, CDCl3)
are reported in δ (ppm) using the δ 7.26 signal of CDCl3, the δ 13.71 (s, 1H), 6.26 (d, J = 2.2 Hz, 1H), 6.24 (d, J = 2.2 Hz, 1H),
δ 3.31 signal of CD3OD or the δ 2.50 signal of DMSO-d6 as an 5.25 (s, 2H), 5.17 (s, 2H), 3.52 (s, 3H), 3.47 (s, 3H), 2.65 (s, 3H).
internal standard. 13C chemical shifts are reported in δ (ppm)
7,8-Bis(benzyloxy)-4-hydroxy-2H-chromen-2-one (10a). To a
using the δ 77.23 signal of CDCl3, the δ 49.15 signal of CD3OD, solution of 1-(3,4-bis(benzyloxy)-2-hydroxyphenyl)ethanone 9a
or the δ 39.51 signal of DMSO-d6 as an internal standard. The (5.30 g, 15 mmol) and diethyl carbonate (3.54 g, 30 mmol) in
purity of final compounds was assessed by the analytical 80 mL toluene was added NaH (2.40 g, 60% in oil, 60 mmol) at
HPLC method and found to be >95%. An Agilent 1200 series 0 °C. The mixture was stirred at 80 °C for 2 h. The solution was
HPLC with a Zorbax SB-C18 (4.6 × 50 mm, 5 μm particle sizes) then cooled to rt and 30 mL 5% aqueous NaOH was added.
reversed-phase column was used for analytical HPLC analyses. After stirring for 10 min, the solution was acidified with
The elution buffer was an A/B gradient, where A = 0.1% 10% aqueous citric acid. The pale precipitate was collected and
HCOOH in H2O and B = CH3OH.
dried under reduced pressure to give 10a (4.6 g, 80%). 1H NMR
1-(3,4-Bis(benzyloxy)-2-hydroxyphenyl)ethanone (9a).25 (300 MHz, CD3OD) δ 7.61 (d, J = 9.0 Hz, 1H), 7.47–7.26
1-(2,3,4-Trihydroxyphenyl)ethanone (6.0 g, 35.7 mmol) was (m, 10H), 7.14 (d, J = 9.0 Hz, 1H), 5.24 (s, 2H), 5.19 (s, 1H),
added to a suspension of benzyl bromide (6.0 g, 35.0 mmol), 5.13 (s, 2H).
K2CO3 (8.0 g, 58.0 mmol) and KI (0.3 g, 1.5 mmol) in DMF
(100 mL). The reaction mixture was heated to 60 °C for 4 h. thetic route as compound 10a starting from 9b–e, respectively.
Upon completion, 200 mL H2O and 300 mL EtOAc were added. 4-Hydroxy-7,8-bis(methoxymethoxy)-2H-chromen-2-one
Compounds 10b–e were prepared utilizing the same syn-
The aqueous phase was extracted with EtOAc and the com- (10b). White solid (1.0 g, 91%). 1H NMR (300 MHz, DMSO-d6)
bined organic phase was washed with water and brine, dried δ 11.60 (br, 1H), 7.47 (d, J = 8.7 Hz, 1H), 7.00 (d, J = 8.7 Hz,
over anhydrous Na2SO4 and concentrated. The residue was 1H), 5.53 (s, 1H), 5.20 (s, 2H), 5.13 (s, 2H), 3.58 (s, 3H), 3.43
purified through column chromatography (eluent, PE–EtOAc = (s, 3H).
4 : 1) to afford 1-(2,3,4-tris(benzyloxy)phenyl)ethanone (15.6 g,
4-Hydroxy-7,8-dimethoxy-2H-chromen-2-one
(10c).28 Pale
100%). Magnesium bromide etherate (5.3 g, 20.5 mmol) was solid. 1H NMR (300 MHz, DMSO-d6) δ 12.41 (s, 1H), 7.54 (d,
added portionwise to a solution of 1-(2,3,4-tris(benzyloxy) J = 9.0 Hz, 1H), 7.09 (d, J = 9.0 Hz, 1H), 5.45 (s, 1H), 3.90 (s, 3H),
phenyl)ethanone (9.0 g, 20.5 mmol) in ether (50 mL). The 3.80 (s, 3H).
mixture was stirred at room temperature for 14 h, and then
4-Hydroxy-5,7-bis(methoxymethoxy)-2H-chromen-2-one
cooled to 0 °C and quenched with 1 M aqueous HCl (100 mL). (10d). Pale solid (100 mg, 30%). 1H NMR (300 MHz, CDCl3)
The aqueous phase was extracted with EtOAc and the com- δ 9.36 (s, 1H), 6.74 (d, J = 2.1 Hz, 1H), 6.71 (d, J = 2.1 Hz, 1H),
bined organic phase was washed with water and brine, dried 5.57 (s, 1H), 5.39 (s, 2H), 5.20 (s, 2H), 3.57 (s, 3H), 3.48 (s, 3H).
over anhydrous Na2SO4 and concentrated. The residue was
4-Hydroxy-2H-chromen-2-one (10e).29 Pale yellow solid. 1H
purified by flash chromatography (PE–EtOAc) to give com- NMR (300 MHz, DMSO-d6) δ 12.48 (s, 1H), 7.82 (d, J = 7.2 Hz,
1
pound 9a (5.3 g, 74%). H NMR (300 MHz, CDCl3) δ 12.62 (s, 1H), 7.67–7.58 (m, 1H), 7.38–7.30 (m, 2H), 5.88 (s, 1H).
H), 7.46–7.28 (m, 11H), 6.49 (d, J = 8.7 Hz, 1H), 5.16 (s, 2H),
5.11(s, 2H), 2.56 (s, 3H).
4,7,8-Trihydroxy-2H-chromen-2-one (2). 10b (2.0 g, 7.1 mmol)
was dissolved in 20 mL 3 N HCl–EtOAc and 0.5 mL MeOH.
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The reaction mixture was stirred at room temperature for was added CH2N2 in Et2O (1 M, 3.4 eq., 0.4 mL) at 0 °C. After
4 h. The white solid was isolated by filtration to afford 2 stirring at 0 °C for 2 h, the reaction was quenched with 0.1 mL
1
(1.3 g, 94.5%). H NMR (300 MHz, DMSO-d6) δ 12.10 (s, 1H), AcOH and concentrated under reduced pressure. The residue
9.97 (br s, 1H), 9.20 (br s, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.77 (d, was purified through column chromatography (eluent,
J = 8.4 Hz, 1H), 5.39 (s, 1H). 13C NMR (75 MHz, DMSO-d6) δ PE–EtOAc = 2 : 1) to afford 3,3′-methylenebis(7,8-bis(benzyloxy)-
166.5, 162.3, 149.7, 143.9, 132.0, 113.3, 111.8, 108.4, 87.8.
3,3′-Methylenebis(4,7,8-trihydroxy-2H-chromen-2-one)
4-methoxy-2H-chromen-2-one) (45 mg, 43%). 1H NMR (300 MHz,
(3). CDCl3) δ 7.52–7.29 (m, 22H), 6.90 (d, J = 8.7 Hz, 2H), 5.18 (s,
To a solution of 2 (500 mg, 2.58 mmol) and paraformaldehyde 8H), 4.06 (s, 6H), 3.93 (s, 2H). 3,3′-methylenebis(7,8-bis(benzyl-
(50 mg, 0.56 mmol) in 10 mL EtOH was added Et3N (0.5 mL). oxy)-4-methoxy-2H-chromen-2-one) (40 mg, 0.0507 mmol) and
The reaction mixture was stirred at room temperature for 24 h. Pd(OH)2 (5 mg) in 10 mL THF were placed under a hydrogen
The resulting pale solid was isolated by filtration, washed with atmosphere (H2, 1 atm.) and stirred for 2 h. The mixture was
10% aqueous citric acid, and dried under vacuum to afford 3 then filtered through a Celite pad. The Celite pad was washed
(460 mg, 89%). Mp: >280 °C. HPLC: 99.37%, tR = 5.408 min. with 4 × 10 mL of MeOH. The filtrate was concentrated and
1H NMR (300 MHz, DMSO-d6) δ 10.01 (br s, 6H), 7.27 (d, J = purified through column chromatography on reverse phase
8.7 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H), 3.71 (s, 2H). 13C NMR C-18 silica gel (eluent, H2O–CH3CN = 3 : 2 to 1 : 1). After lyophili-
(75 MHz, DMSO-d6) δ 165.8, 163.3, 149.4, 142.3, 132.0, 113.5, zation, 4 was obtained as a yellowish brown solid (12 mg,
112.5, 109.0, 99.5, 18.9. HRMS (ESI): calcd for C19H12O10Na 55.3%). Mp: 262–264 °C. HPLC: 98.28%, tR = 4.005 min.
[M + Na]+, 423.0328. Found: [M + Na]+, 423.0321.
Compounds 11a–b, 5 (ref. 28) and 8 (ref. 30) were prepared 8.4 Hz, 2H), 4.00 (s, 6H), 3.87 (s, 2H). 13C NMR (75 MHz,
utilizing the same synthetic route as compound 3 starting CD3OD CDCl3) 167.2, 165.7, 150.4, 143.8, 133.6,
115.2, 113.5, 112.3, 111.3, 62.7, 21.2. HRMS (ESI): calcd
1H NMR (300 MHz, CD3OD) δ 7.13 (d, J = 8.7 Hz, 2H), 6.82 (d, J =
+
δ
from 10a,c–e.
3,3′-Methylenebis(7,8-bis(benzyloxy)-4-hydroxy-2H-chromen- for C21H16O10Na [M + Na]+, 451.0641. Found: [M + Na]+,
2-one) (11a). White solid (80 mg, 79%). 1H NMR (300 MHz, 451.0637.
CDCl3) δ 7.75 (d, J = 9.0 Hz, 2H), 7.47–7.26 (m, 20H), 6.89 (d,
J = 8.7 Hz, 2H), 5.15 (s, 4H), 5.12 (s, 4H), 3.83 (s, 2H).
Diethyl 2,4-diacetylpentanedioate (13).31
Et2NH (412 μL, 4.0 mmol) and CH2Br2 (2.1 mL, 30.0 mmol)
A
mixture of
3,3′-Methylenebis(4-hydroxy-5,7-bis(methoxymethoxy)-2H- was heated to 50 °C for 1.5 h and then cooled to rt. The
chromen-2-one) (11b). White solid (54 mg, 74%). 1H NMR mixture was added to a solution of ethyl acetoacetate (258 mg,
(300 MHz, CDCl3) δ 10.55 (s, 2H), 6.72 (d, J = 2.1 Hz, 2H), 6.71 2.0 mmol) in 8 mL of CH2Cl2 and stirred at rt. Upon com-
(d, J = 2.1 Hz, 2H), 5.31 (s, 4H), 5.19 (s, 4H), 3.79 (s, 2H), 3.55 pletion (2 h), the reaction mixture was concentrated, and the
(s, 6H), 3.47 (s, 6H).
crude mixture was purified by column chromatography on
3,3′-Methylenebis(4,5,7-trihydroxy-2H-chromen-2-one)
Compound 11b (50 mg, 0.0867 mmol) was dissolved in 3 mL less oil (123 mg, 45%). H NMR (300 MHz, CDCl3) δ 4.27–4.13
2 N HCl–EtOAc and stirred at room temperature for 1 h. The (m, 4H), 3.53 (t, J = 7.2 Hz, 1H), 2.46–2.29 (m, 2H), 2.26 (s, 6H),
(7). silica gel (EtOAc–PE) to give the desired product 13 as a color-
1
reaction mixture was evaporated to afford 7 as a brown solid 1.34–1.22 (m, 6H).
1
(27 mg, 80%). Mp: >280 °C. HPLC: 95.51%, tR = 3.911 min. H
3,3′-Methylenebis(7,8-dihydroxy-4-methyl-2H-chromen-2-one) (6).
NMR (300 MHz, DMSO-d6) δ 10.03 (s, 2H), 6.05 (d, J = 2.1 Hz, To a mixture of pyrogallol 12 (500 mg, 4 mmol) and
2H), 5.99 (d, J = 1.5 Hz, 2H), 3.45 (s, 2H). 13C NMR (75 MHz, diethyl 2,4-diacetylpentanedioate 13 (544 mg, 2 mmol) at 0 °C
DMSO-d6) δ 169.1, 163.1, 160.7, 158.0, 154.6, 98.3, 98.2, 97.6, was added 70% H2SO4. The reaction mixture was stirred at
93.4, 17.7. HRMS (ESI): calcd for C19H12O10Na [M + Na]+, room temperature for 30 min, and then poured into water
423.0328. Found: [M + Na]+, 423.0316.
(50 mL). The tan solid was isolated by filtration and dried
3,3′-Methylenebis(4-hydroxy-7,8-dimethoxy-2H-chromen-2-one) (5). under vacuum to afford 6 (50 mg, 5%). Mp: >280 °C. HPLC:
1
Pale solid (100 mg, 86%). Mp: 286–287 °C. HPLC: 95.58%, 96.23%, tR = 2.799 min. H NMR (300 MHz, DMSO-d6) δ 7.12
tR = 7.945 min. 1H NMR (300 MHz, DMSO-d6) δ 7.63 (d, J = (d, J = 7.2 Hz, 1H), 6.79 (d, J = 7.2 Hz, 1H), 3.87 (s, 2H), 2.41
8.7 Hz, 2H), 7.09 (d, J = 9.0 Hz, 2H), 3.89 (s, 6H), 3.78 (s, 6H), (s, 6H). HRMS (ESI): calcd for C21H16O8Na [M + Na]+, 419.0743.
3.71 (s, 2H). HRMS (ESI): calcd for C23H21O10 [M + H]+, Found: [M + Na]+, 419.0735.
457.1135. Found: [M + H]+, 457.1119.
3,4-Bis(benzyloxy)-2-hydroxybenzaldehyde
3,3′-Methylenebis(4-hydroxy-2H-chromen-2-one) (8). White pound 24 was prepared utilizing the same synthetic route as
(24).32 Com-
solid (400 mg, 90%). Mp: 264–265 °C. HPLC: 97.52%, tR
=
compound 9a starting from 2,3,4-trihydroxybenzaldehyde.
11.516 min. 1H NMR (300 MHz, DMSO-d6) δ 7.92 (d, J = 7.8 Hz, White solid (2.4 g, 84%). 1H NMR (300 MHz, CDCl3) δ 11.24 (s,
2H), 7.58 (t, J = 7.5 Hz, 2H), 7.41–7.27 (m, 4H), 3.79 (s, 2H). 1H), 9.74 (s, 1H), 7.50–7.29 (m, 10H), 7.24 (d, J = 8.7 Hz, 1H),
13C NMR (75 MHz, DMSO-d6)
131.7, 123.9, 123.4, 116.9, 116.1, 102.3, 19.4. HRMS (ESI): calcd
for C19H12O6Na [M + Na]+, 459.0532. Found: [M + Na]+, pared utilizing the same synthetic route as compound 9d start-
δ
163.7, 162.7, 151.9, 6.62 (d, J = 8.4 Hz, 1H), 5.18 (s, 2H), 5.13 (s, 2H).
Compounds 19 (ref. 25) and 56a–b (ref. 33,34) were pre-
459.0521.
ing from different salicylaldehydes.
3,3′-Methylenebis(7,8-dihydroxy-4-methoxy-2H-chromen-2-one) (4).
To a solution of 11a (90 mg, 0.118 mmol) in 6 mL THF solid (2.26 g, 29%). H NMR (300 MHz, CDCl3) δ 11.29 (s, 1H),
2-Hydroxy-3,4-bis(methoxymethoxy)benzaldehyde (19). White
1
3726 | Org. Biomol. Chem., 2014, 12, 3721–3734
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9.76 (s, 1H), 7.28 (d, J = 8.7 Hz, 1H), 6.91 (d, J = 8.7 Hz, 1H),
5.30 (s, 2H), 5.20 (s, 2H), 3.64 (s, 3H), 3.51 (s, 3H).
7-(Methoxymethoxy)-2-oxo-2H-chromene-3-carboxylic
(59b). White solid (237 mg, 76%). H NMR (300 MHz, CDCl3)
acid
1
2-Hydroxy-4-(methoxymethoxy)benzaldehyde (56a). White δ 8.76 (s, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.13–7.06 (m, 2H), 5.30
1
solid (4.0 g, 61%). H NMR (300 MHz, CDCl3) δ 11.36 (s, 1H), (s, 2H), 3.51 (s, 3H).
9.74 (s, 1H), 7.45 (d, J = 8.4 Hz, 1H), 6.65 (dd, J = 8.4, 2.4 Hz,
1H), 6.60 (d, J = 2.4 Hz, 1H), 5.22 (s, 2H), 3.48 (s, 3H).
7-(Methoxymethoxy)-2-oxo-2H-chromene-3-carboxylic
acid
(59c). Yellowish-white solid (367 mg, 67%). 1H NMR
2-Hydroxy-3-(methoxymethoxy)benzaldehyde (56b). White (300 MHz, CDCl3) δ 8.85 (s, 1H), 7.47 (d, J = 8.7 Hz, 1H), 7.06
solid (300 mg, 15%). 1H NMR (300 MHz, CDCl3) δ 11.12 (s, (d, J = 8.7 Hz, 1H), 4.04 (s, 3H), 4.02 (s, 3H).
1H), 9.91 (s, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.26 (dd, J = 7.8, 1.8
Hz, 1H), 6.96 (t, J = 7.8 Hz, 1H), 5.26 (s, 3H), 3.53 (s, 3H).
3-Amino-7,8-bis(methoxymethoxy)-2H-chromen-2-one (21).
To mixture of 2-hydroxy-3,4-bis(methoxymethoxy)benz-
a
2-Hydroxy-3,4-dimethoxybenzaldehyde (56c).35 To a solution aldehyde 19 (2.26 g, 8.80 mmol) and ethyl nitroacetate (1.40 g,
of 2,3,4-trihydroxybenzaldehyde (2.1 g, 13.6 mmol) and K2CO3 10.6 mmol) in 60 mL dry benzene was added piperidine
(6.6 g, 47.7 mmol) in 50 mL acetone was added CH3I (3.0 mL, (174 μL, 1.76 mmol). The reaction mixture was heated to reflux
47.7 mmol). The reaction mixture was stirred at 60 °C for 24 h, for 6 h with a Dean–Stark trap to collect the water. The reaction
and then cooled to rt. The residue was diluted with 100 mL was then cooled to rt and purified by flash chromatography
EtOAc and 50 mL H2O. The aqueous phase was extracted with (eluent, CH2Cl2) to give 7,8-bis(methoxymethoxy)-3-nitro-2H-
EtOAc. The combined organic phase was washed with water chromen-2-one as a yellow solid (1.90 g, 65%). 1H NMR
and brine, dried over anhydrous Na2SO4 and concentrated (300 MHz, CDCl3) δ 8.74 (s, 1H), 7.46 (d, J = 8.7 Hz, 1H), 7.28
under reduced pressure to give 2,3,4-trimethoxybenzaldehyde (d, J = 8.7 Hz, 1H), 5.37 (s, 2H), 5.25 (s, 2H), 3.71 (s, 3H), 3.55
1
(2.6 g, 97.2%). H NMR (300 MHz, CDCl3) δ 10.23 (s, 1H), 7.59 (s, 3H). 7,8-Bis(methoxymethoxy)-3-nitro-2H-chromen-2-one
(d, J = 9.0 Hz, 1H), 6.74 (d, J = 9.0 Hz, 1H), 4.02 (s, 3H), 3.92 (350 mg, 1.07 mmol) and 10% Pd/C (14 mg) in 15 mL EtOAc
(s, 3H), 3.87 (s, 3H). To a solution of 2,3,4-trimethoxybenzalde- was stirred under hydrogen (H2, 1 atm.) for 2 h. The mixture
hyde (546 mg, 2.78 mmol) in 20 mL benzene was added anhy- was collected through a Celite pad. The Celite pad was washed
drous AlCl3 (408 mg, 3.06 mmol). After stirring for 5 min at with 4 × 20 mL of EtOAc. The filtrate was concentrated and
room temperature, the mixture was heated to 80 °C for 6 h, purified by flash chromatography column (PE–EtOAc) to
and then cooled to rt. 30 mL ice water and 3 mL concentrated provide aminocoumarin 21 as a yellow solid (190 mg, 63%).
HCl were added with stirring. The aqueous phase was 1H NMR (300 MHz, CDCl3) δ 7.05 (d, J = 8.7 Hz, 1H), 6.96 (d,
extracted with EtOAc. The combined organic phase was J = 8.7 Hz, 1H), 6.65 (s, 1H), 5.24 (s, 2H), 5.22 (s, 2H), 4.13 (br,
washed with water and brine, dried over anhydrous Na2SO4 2H), 3.70 (s, 3H), 3.51 (s, 3H).
and concentrated. The residue was purified through column
N-(7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)-7,8-dihydroxy-2-oxo-
(14). Acid 20 (55 mg,
chromatography (eluent, PE–EtOAc = 10 : 1 to 8 : 1) to give 56c 2H-chromene-3-carboxamide
1
as a white solid (398 mg, 78.6%). H NMR (300 MHz, CDCl3) 0.177 mmol), aminocoumarin 21 (50 mg, 0.177 mmol) and
δ 11.21 (s, 1H), 9.75 (s, 1H), 7.29 (d, J = 8.7 Hz, 1H), 6.61 (d, J = EDCI (52 mg, 0.267 mmol) were dissolved in 3 mL of 30% pyri-
8.7 Hz, 1H), 3.95 (s, 3H), 3.90 (s, 3H).
dine/CH2Cl2 and stirred at rt for 2 h. The solvent was evapor-
General procedures for the preparation of acidcoumarins ated and the residue was purified by column chromatography
(20, 25, 59a–c). A mixture of the corresponding salicylalde- (CH2Cl2–acetone = 50 : 1 to 40 : 1) to give N-(7,8-bis(methoxy-
hydes 19, 24 or 56a–c (2 mmol), isopropylidene malonate methoxy)-2-oxo-2H-chromen-3-yl)-7,8-bis(methoxymethoxy)-
(2.4 mmol) and piperidinium acetate (0.1 mmol) in 30 mL 2-oxo-2H-chromene-3-carboxamide (17 mg, 17%). 1H NMR
anhydrous ethanol was heated to 60 °C for 24 h. Then (300 MHz, CDCl3) δ 11.46 (s, 1H), 8.86 (s, 1H), 8.79 (s, 1H),
the mixture was cooled to 0 °C and the precipitate was 7.45 (d, J = 8.7 Hz, 1H), 7.26–7.21 (m, 2H), 7.14 (d, J = 8.7 Hz,
collected by filtration, washed with 5 mL cold ethanol and 1H), 5.34 (s, 2H), 5.28 (s, 2H), 5.26 (s, 2H), 5.26 (s, 2H), 3.74 (s,
dried in vacuo to afford the corresponding acidcoumarins 3H), 3.72 (s, 3H), 3.53 (s, 3H), 3.52 (s, 3H). This intermediate
(20, 25, 59a–c).
(17 mg, 0.0296 mmol) was dissolved in 3 mL 2 M HCl in EtOAc
7,8-Bis(methoxymethoxy)-2-oxo-2H-chromene-3-carboxylic and stirred at rt for 2 h. The yellow solid was isolated by fil-
acid (20). White solid (780 mg, 60%). 1H NMR (300 MHz, tration to afford 14 (10 mg, 85%). Mp: >280 °C. HPLC: 95.35%,
1
CDCl3) δ 12.25 (br, 1H), 8.85 (s, 1H), 7.46 (d, J = 8.7 Hz, 1H), tR = 2.868 min. H NMR (300 MHz, DMSO-d6) δ 11.22 (s, 1H),
7.29 (d, J = 8.7 Hz, 1H), 5.36 (s, 2H), 5.26 (s, 2H), 3.71 (s, 3H), 10.80 (br s, 1H), 9.98 (br s, 1H), 9.69 (br s, 1H), 9.42 (br s, 1H),
3.53 (s, 3H).
8.89 (s, 1H), 8.73 (s, 1H), 7.41 (d, J = 8.7 Hz, 1H), 7.06 (d, J =
7,8-Bis(benzyloxy)-2-oxo-2H-chromene-3-carboxylic
acid 8.7 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H).
(25). White solid (550 mg, 46%). 1H NMR (300 MHz, CDCl3) 13C NMR (75 MHz, DMSO-d6) δ 161.3, 160.6, 158.0, 152.9,
δ 12.23 (br, 1H), 8.82 (s, 1H), 7.47–7.38 (m, 8H), 7.36–7.28 (m, 149.6, 148.1, 144.3, 139.9, 132.2, 132.0, 125.1, 122.0, 120.7,
3H), 7.08 (d, J = 8.7 Hz, 1H), 5.27 (s, 2H), 5.21 (s, 2H).
118.2, 113.8, 113.2, 112.3, 112.2, 112.0. HRMS (ESI): calcd for
8-(Methoxymethoxy)-2-oxo-2H-chromene-3-carboxylic
acid C19H11NO9Na [M + Na]+, 420.0332. Found: [M + Na]+, 420.0312.
1
(59a). Beige solid (145 mg, 35%). H NMR (300 MHz, CDCl3)
trans-N1,N2-Bis(7,8-dihydroxy-2-oxo-2H-chromen-3-yl)cyclo-
δ 8.93 (s, 1H), 7.59 (t, J = 4.8 Hz, 2H), 7.38 (d, J = 4.5 Hz, 2H), hexane-1,2-dicarboxamide (16). trans-1,2-Cyclohexanedicar-
5.35 (s, 2H), 3.56 (s, 3H).
boxylic acid 23 (26 mg, 0.151 mmol), aminocoumarin 21
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(85 mg, 0.302 mmol) and EDCI (87 mg, 0.453 mmol) were dis- 2 mL CH3OH and 2 mL EtOAc were hydrogenated (H2, 1 atm.)
solved in 3 mL of 30% pyridine/CH2Cl2. The mixture was for 4 h. The mixture was filtered through a Celite pad. The
heated to 50 °C for 40 h. The solvent was evaporated and the Celite pad was washed with 4 × 10 mL of MeOH. The filtrate
residue was purified by column chromatography (PE–EtOAc) was concentrated and purified by flash chromatography
to give trans-N1,N2-bis(7,8-bis(methoxymethoxy)-2-oxo-2H- column (eluent, CH2Cl2–CH3OH = 20 : 1) to give 3-(4-(7,8-bis-
chromen-3-yl)cyclohexane-1,2-dicarboxamide (25 mg, 23.7%). (methoxymethoxy)-2-oxo-2H-chromene-3-carbonyl)piperazine-
1H NMR (300 MHz, CDCl3) δ 8.58 (s, 2H), 8.12 (s, 2H), 7.14 1-carbonyl)-7,8-dihydroxy-2H-chromen-2-one (27 mg, 90%).
(d, J = 8.9 Hz, 2H), 7.09 (d, J = 8.1 Hz, 2H), 5.24 (s, 4H), 5.22 This intermediate was dissolved in 2 mL DCM and 3 drops of
(s, 4H), 3.69 (s, 6H), 3.49 (s, 6H), 2.81–2.69 (m, 2H), 2.14–2.02 MeOH, then 2 mL of 2 M HCl in EtOAc was added. The
(m, 2H), 1.95–1.85 (m, 2H), 1.65–1.50 (m, 2H), 1.45–1.35 (m, mixture was stirred at room temperature for 2 h, then the
2H). LS-MS: m/z: 721.2 [M + Na]+. This intermediate (25 mg, yellow solid was isolated by filtration to afford 17 (21 mg,
0.0358 mmol) was dissolved in 3 mL of 2 M HCl in EtOAc and 91%). Mp: >280 °C. HPLC: 97.06%, tR = 2.825 min. 1H NMR
stirred at rt for 2 h. The mixture was concentrated and purified (300 MHz, DMSO-d6) δ 10.31 (s, 2H), 9.45 (br s, 2H), 8.08 (s,
by flash chromatography (CH2Cl2–CH3OH = 20 : 1) to provide 2H), 7.10 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 3.66–3.38
16 as a yellow solid (14 mg, 75%). Mp: 284–285 °C. HPLC: (m, 8H). 13C NMR (75 MHz, DMSO-d6) δ 163.8, 158.0, 150.6,
95.07%, tR = 2.648 min. 1H NMR (300 MHz, CD3OD) δ 8.45 144.0, 143.7, 132.0, 119.7, 119.2, 113.0, 111.5, 46.7, 46.1, 41.7,
(s, 2H), 6.88 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.4 Hz, 2H), 41.2. HRMS (ESI): calcd for C24H18N2O10Na [M + Na]+,
2.94–2.86 (m, 2H), 2.14–2.04 (m, 2H), 1.94–1.80 (m, 2H), 517.0859. Found: [M + Na]+, 517.3680.
1.62–1.38 (m, 4H). LS-MS: m/z: 523.1 [M + H]+, 545.1 [M + Na]+.
(S)-2-Amino-N-(7,8-bis(methoxymethoxy)-2-oxo-2H-chromen-
cis-N1,N2-Bis(7,8-dihydroxy-2-oxo-2H-chromen-3-yl)cyclo- 3-yl)-4-methylpentanamide (29). Fmoc-Leu-OH 28 (227 mg,
hexane-1,2-dicarboxamide (15). This compound was prepared 0.641 mmol), aminocoumarin 21 (120 mg, 0.427 mmol) and
utilizing the same synthetic route as compound 16 starting EDCI (164 mg, 0.854 mmol) were dissolved in 5 mL of 30%
from cis-1,2-cyclohexanedicarboxylic acid 22 and aminocou- pyridine/CH2Cl2. The mixture was stirred at rt for 36 h. The
marin 21. Yellow solid (3 mg, 3%). Mp = 216–218 °C. HPLC: solvent was evaporated and the residue was purified by
1
95.33%, tR = 2.851 min. H NMR (300 MHz, CD3OD) δ 8.47 (s, column chromatography (PE–EtOAc) to give (R)-(9H-fluoren-
2H), 6.90 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 3.04 (s, 9-yl)methyl (1-((7,8-bis(methoxymethoxy)-2-oxo-2H-chromen-3-yl)-
2H), 2.32–2.20 (m, 2H), 1.92–1.76 (m, 4H), 1.62–1.46 (m, 2H). amino)-4-methyl-1-oxopentan-2-yl)carbamate as a white solid
LS-MS: m/z: 523.2 [M + H]+, 545.2 [M + Na]+.
(190 mg, 72.1%). 1H NMR (300 MHz, CDCl3) δ 8.60 (s, 1H),
7,8-Bis(benzyloxy)-3-(piperazine-1-carbonyl)-2H-chromen- 8.57 (br, 1H), 7.75 (d, J = 7.5 Hz, 2H), 7.62–7.56 (m 2H), 7.38 (t,
2-one (26). A mixture of 25 (100 mg, 0.249 mmol), 1-Boc-piper- J = 7.5 Hz, 2H), 7.30 (t, J = 7.2 Hz, 2H), 7.18 (d, J = 8.7 Hz, 1H),
azine (46 mg, 0.249 mmol), EDCI (72 mg, 0.373 mmol) and 7.13 (d, J = 8.7 Hz, 1H), 5.32–5.18 (m, 5H), 4.48 (d, J = 6.0 Hz,
DMAP (6 mg, 0.049 mmol) in 10 mL CH2Cl2 was stirred at 2H), 4.38 (s, 1H), 4.23 (t, J = 6.6 Hz, 1H), 3.70 (s, 3H), 3.52 (s,
room temperature for 4 h and then concentrated. The residue 3H), 1.78–1.54 (m, 3H), 0.96 (s, 6H). To a solution of this inter-
was purified by flash chromatography column (PE–EtOAc) to mediate (190 mg, 0.308 mmol) in 10 mL acetonitrile was
give tert-butyl 4-(7,8-bis(benzyloxy)-2-oxo-2H-chromene-3-car- added piperidine (31 μL, 0.308 mmol). The reaction mixture
bonyl)piperazine-1-carboxylate (130 mg, 91.7%). 1H NMR was stirred at rt for 6 h. The solvent was evaporated and the
(300 MHz, CDCl3) δ 7.87 (s, 1H), 7.51–7.27 (m, 10H), 7.19 (d, residue was purified by column chromatography (PE–EtOAc =
J = 8.7 Hz, 1H), 6.94 (d, J = 8.7 Hz, 1H), 5.21 (s, 2H), 5.20 (s, 2 : 1 to 1 : 1) to give 29 as a white solid (85 mg, 70.1%).
2H), 3.73 (s, 2H), 3.57–3.47 (m, 4H), 3.34 (s, 2H), 1.47 (s, 9H). 1H NMR (300 MHz, CDCl3) δ 10.10 (s, 1H), 8.65 (s, 1H), 7.18 (d,
This intermediate was dissolved in 3 mL of 2 M HCl in EtOAc J = 8.7 Hz, 1H), 7.11 (d, J = 8.7 Hz, 1H), 5.26 (s, 2H), 5.24 (s,
at 0 °C and then warmed to rt. After stirring at room tempera- 2H), 3.70 (s, 3H), 3.60–3.53 (m, 1H), 3.51 (s, 3H), 1.92–1.63 (m,
ture for 1 h, the mixture was concentrated to give 26 as its 5H), 1.06–0.88 (m, 6H).
hydrochloride salt.
(S)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-
7,8-Bis(benzyloxy)-3-(4-(7,8-bis(methoxymethoxy)-2-oxo-2H- 4-methyl-1-oxopentan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-3-
chromene-3-carbonyl)piperazine-1-carbonyl)-2H-chromen-2-one carboxamide (18). Compound 29 (85 mg, 0.216 mmol), 20
(27). To a mixture of 26 hydrochloride (0.228 mmol), 20 (80 mg, 0.259 mmol), EDCI (62 mg, 0.324 mmol) and DMAP
(85 mg, 0.274 mmol), EDCI (66 mg, 0.342 mmol) and DMAP (6 mg, 0.049 mmol) were dissolved in 10 mL CH2Cl2 and
(5 mg, 0.041 mmol) in 10 mL CH2Cl2 was added Et3N (95 μL, stirred at rt for 2 h. The solvent was evaporated and the
0.684 mmol). The reaction mixture was stirred at room temp- residue was purified by column chromatography (PE–EtOAc =
erature overnight, and then concentrated and purified by flash 2 : 1 to 1 : 1) to give (R)-N-(1-((7,8-bis(methoxymethoxy)-2-oxo-
chromatography column (PE–acetone) give 27 (70 mg, 40%). 2H-chromen-3-yl)amino)-4-methyl-1-oxopentan-2-yl)-7,8-bis-
1H NMR (300 MHz, CDCl3) δ 7.92 (s, 1H), 7.89 (s, 1H), (methoxymethoxy)-2-oxo-2H-chromene-3-carboxamide (120 mg,
7.54–7.07 (m, 13H), 6.95 (d, J = 8.4 Hz, 1H), 5.32–5.15 (m, 8H), 81%). 1H NMR (300 MHz, CDCl3) δ 9.15 (d, J = 6.9 Hz, 1H), 8.88
3.87 (s, 4H), 3.69 (s, 3H), 3.51 (s, 4H), 3.48 (s, 3H).
(s, 1H), 8.87 (s, 1H), 8.63 (s, 1H), 7.40 (d, J = 9.0 Hz, 1H), 7.21
3,3′-(Piperazine-1,4-dicarbonyl)bis(7,8-dihydroxy-2H-chromen- (d, J = 9.0 Hz, 1H), 7.17 (d, J = 8.7 Hz, 1H), 7.11 (d, J = 8.7 Hz, 1H),
2-one) (17). 27 (40 mg, 0.0524 mmol) and 10% Pd/C (5 mg) in 5.33 (s, 2H), 5.25 (s, 4H), 5.22 (s, 2H), 4.78–4.70 (m, 1H), 3.71 (s, 3H),
3728 | Org. Biomol. Chem., 2014, 12, 3721–3734
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3.68 (s, 3H), 3.52 (s, 3H), 3.50 (s, 3H), 1.95–1.75 (m, 3H), 9.43–9.20 (br s, 1H), 9.10 (d, J = 7.8 Hz, 1H), 8.77 (s, 1H), 8.47
1.02 (d, J = 6.0 Hz, 3H), 0.98 (d, J = 6.3 Hz, 3H). This intermedi- (s, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.91 (d,
ate (140 mg, 0.175 mmol) was dissolved in 2 mL DCM and J = 8.7 Hz, 1H), 6.81 (d, J = 8.7 Hz, 1H), 5.02–4.93 (m, 1H),
2 mL of 2 M HCl in EtOAc, and stirred at rt for 2 h. The yellow 1.90–1.65 (m, 2H), 1.45–1.30 (m, 2H), 0.91 (t, J = 7.2 Hz, 3H).
solid was isolated by filtration to afford 18 (96 mg, 92%). Mp: 13C NMR (75 MHz, DMSO-d6) δ 171.5, 161.3, 161.1, 157.7,
180–184 °C. HPLC: 97.97%, tR
=
3.511 min. 1H NMR 152.3, 149.0, 148.1, 144.2, 140.3, 132.0, 131.9, 127.7, 121.6,
(300 MHz, DMSO-d6) δ 10.66 (br s, 1H), 9.92 (s, 2H), 9.59 (br s, 120.2, 118.1, 113.6, 113.1, 112.8, 112.1, 111.8, 53.0, 34.8, 18.3,
1H), 9.32 (br s, 1H), 9.04 (d, J = 8.1 Hz, 1H), 8.77 (s, 1H), 8.46 13.7. HRMS (ESI): calcd for C21H14N2O10Na [M + Na]+,
(s, 1H), 7.33 (d, J = 8.7 Hz, 1H), 7.00 (d, J = 8.7 Hz, 1H), 6.91 (d, 519.1016. Found: [M + Na]+, 519.1012.
J = 8.7 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 5.04–4.96 (m, 1H),
1.68–1.66 (m, 3H), 0.94 (d, J = 5.1 Hz, 6H). 13C NMR (75 MHz, 1-oxobutan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-3-carboxamide
DMSO-d6) δ 171.8, 161.4, 161.1, 157.7, 152.3, 149.1, 148.1, (33). Yellow solid. Mp: 179–183 °C. HPLC: 96.66%, tR
(S)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-3-methyl-
=
1
144.2, 140.3, 132.0, 131.9, 127.8, 121.6, 120.2, 118.1, 113.6, 2.653 min. H NMR (300 MHz, DMSO-d6) δ 10.65 (s, 1H), 9.94
113.1, 112.7, 112.1, 111.8, 52.0, 41.4, 24.6, 23.1, 21.8. HRMS (s, 1H), 9.90 (s, 1H), 9.60 (s, 1H), 9.31 (s, 1H), 9.13 (d, J =
(ESI): calcd for C25H22N2O10Na [M + Na]+, 533.1172. Found: [M 8.7 Hz, 1H), 8.78 (s, 1H), 8.46 (s, 1H), 7.34 (d, J = 8.7 Hz, 1H),
+ Na]+, 533.1167.
7.00 (d, J = 8.7 Hz, 1H), 6.91 (d, J = 8.7 Hz, 1H), 6.81 (d, J =
3-Amino-7-(methoxymethoxy)-2H-chromen-2-one (57a). This 8.4 Hz, 1H), 4.97–4.90 (m, 1H), 2.27–2.08 (m, 1H), 1.05–0.85
compound was prepared utilizing the same synthetic route as (m, 6H). 13C NMR (75 MHz, DMSO-d6) δ 170.89, 161.5, 161.2,
compound 21, starting from 2-hydroxy-4-(methoxymethoxy) 157.7, 152.3, 149.1, 148.1, 144.2, 140.3, 132.0, 131.9, 127.9,
benzaldehyde 56a. Yellow solid (780 mg, 36%). 1H NMR 121.5, 120.1, 118.1, 113.6, 113.0, 112.8, 112.1, 111.9, 57.7, 31.3,
(300 MHz, CDCl3) δ 7.19 (d, J = 8.7 Hz, 1H), 6.99 (d, J = 2.4 Hz, 19.2, 17.4. HRMS (ESI): calcd for C21H14N2O10Na [M + Na]+,
1H), 6.92 (dd, J = 8.4, 2.4 Hz, 1H), 6.68 (s, 1H), 5.18 (s, 2H), 519.1016. Found: [M + Na]+, 519.1003.
3.48 (s, 3H).
Compounds 30–47, 49a, 50a and 51–55 were prepared utiliz- hexan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-3-carboxamide
ing the same synthetic route as compound 18 starting from the (34). Yellow solid. Mp: 246–250 °C. HPLC: 97.35%, tR
(S)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-1-oxo-
=
1
appropriate Fmoc-amino acids, 57a (or 21), and 20 (or 59a–c).
N-(2-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-2-oxoethyl)- (d, J = 7.5 Hz, 1H), 8.77 (s, 1H), 8.47 (s, 1H), 7.33 (d, J = 8.4 Hz,
7,8-dihydroxy-2-oxo-2H-chromene-3-carboxamide (30). Yellow 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.81 (d, J =
3.667 min. H NMR (300 MHz, DMSO-d6) δ 9.88 (s, 1H), 9.11
solid. Mp: >280 °C. HPLC: 95.91%, tR = 2.772 min. 1H NMR 8.4 Hz, 1H), 5.00–4.91 (m, 1H), 1.90–1.65 (m, 2H), 1.40–1.20 (s,
(300 MHz, DMSO-d6) δ 10.65 (s, 1H), 9.94 (s, 1H), 9.78 (s, 1H), 4H), 0.86 (t, J = 6.3 Hz, 3H). 13C NMR (75 MHz, DMSO-d6)
9.60 (s, 1H), 9.32 (s, 1H), 9.15 (t, J = 5.4 Hz, 1H), 8.78 (s, 1H), δ 171.5, 161.4, 161.1, 157.7, 152.3, 149.0, 148.2, 144.3, 140.3,
8.50 (s, 1H), 7.35 (d, J = 8.7 Hz, 1H), 6.99 (d, J = 8.7 Hz, 1H), 132.1, 131.9, 127.7, 121.6, 120.2, 118.1, 113.6, 113.1, 112.8,
6.91 (d, J = 8.7 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 4.28 (d, J = 112.1, 111.9, 53.2, 40.3, 40.1, 39.8, 39.5, 39.2, 39.0, 38.7, 32.4,
5.1 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 168.6, 161.9, 27.1, 21.9, 13.8. HRMS (ESI): calcd for
160.8, 157.7, 152.3, 149.0, 148.0, 144.3, 140.1, 132.1, 131.9, [M + Na]+, 533.1172. Found: [M + Na]+, 533.1166.
126.5, 121.6, 120.4, 118.0, 113.6, 113.1, 112.8, 112.1, 111.8, (R)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-1-oxo-
43.6. HRMS (ESI): calcd for C21H14N2O10Na [M + Na]+, butan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-3-carboxamide
C25H22N2O10Na
477.0546. Found: [M + Na]+, 477.0543.
(35). Yellow solid. Mp: >280 °C. HPLC: 98.61%, tR =
(S)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-1-oxo- 2.294 min. 1H NMR (300 MHz, DMSO-d6) δ 10.66 (br s, 1H),
propan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-3-carboxamide 9.93 (br s, 1H), 9.88 (s, 1H), 9.61 (br s, 1H), 9.33 (br s, 1H), 9.13
(31). Yellow solid. Mp: >280 °C. HPLC: 97.36%, tR
=
(d, J = 7.5 Hz, 1H), 8.77 (s, 1H), 8.48 (s, 1H), 7.34 (d, J = 8.7 Hz,
1
2.043 min. H NMR (300 MHz, DMSO-d6) δ 10.66 (s, 1H), 9.96 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.81 (d, J =
(s, 1H), 9.86 (s, 1H), 9.61 (s, 1H), 9.33 (s, 1H), 9.16 (d, J = 7.2 8.4 Hz, 1H), 5.03–4.83 (m, 1H), 1.94–1.70 (m, 2H), 0.93 (t, J =
Hz, 1H), 8.77 (s, 1H), 8.49 (s, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.01 7.5 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 171.3, 161.4,
(d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.7 Hz, 1H), 6.81 (d, J = 8.7 Hz, 161.1, 157.7, 152.3, 149.0, 148.1, 144.2, 140.3, 132.0, 131.9,
1H), 5.03–4.86 (m, 1H), 1.42 (d, J = 6.9 Hz, 3H). 13C NMR 127.7, 121.6, 120.1, 118.1, 113.6, 113.1, 112.8, 112.1, 111.8,
(75 MHz, DMSO-d6) δ 172.0, 161.1, 161.0, 157.7, 152.3, 149.0, 54.2, 25.9, 9.6. HRMS (ESI): calcd for C23H18N2O10Na
148.1, 144.3, 140.2, 132.1, 131.9, 127.5, 121.6, 120.2, 118.1, [M + Na]+, 505.0859. Found: [M + Na]+, 505.0850.
113.6, 113.1, 112.7, 112.1, 111.8, 49.1, 18.9. HRMS (ESI): calcd
for C22H16N2O10Na [M + Na]+, 497.0703. Found: [M + Na]+, pentan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-3-carboxamide
497.0695. (36). Yellow solid. Mp: >280 °C. HPLC: 95.90%, tR
(R)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-1-oxo-
=
1
(S)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-1-oxo- 2.843 min. H NMR (300 MHz, DMSO-d6) δ 9.89 (s, 1H), 9.11
pentan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-3-carboxamide (d, J = 8.1 Hz, 1H), 8.77 (s, 1H), 8.47 (s, 1H), 7.33 (d, J = 8.7 Hz,
(32). Yellow solid. Mp: >280 °C. HPLC: 98.37%, tR
=
1H), 7.00 (d, J = 8.7 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.81 (d, J =
2.807 min. 1H NMR (300 MHz, DMSO-d6) δ 10.65 (br s, 1H), 8.1 Hz, 1H), 5.08–4.88 (m, 1H), 1.89–1.62 (m, 2H), 1.47–1.27
10.10–9.75 (br s, 1H), 9.89 (s, 1H), 9.70–9.44 (br s, 1H), (m, 2H), 0.91 (t, J = 7.5 Hz, 3H). 13C NMR (75 MHz, DMSO-d6)
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δ 171.5, 161.4, 161.1, 157.7, 152.3, 149.0, 148.1, 144.2, 140.3, 31.0, 28.3, 24.8, 21.9, 13.8. HRMS (ESI): calcd for
132.1, 131.9, 127.7, 121.6, 120.2, 118.1, 113.6, 113.1, 112.8, C27H26N2O10Na [M + Na]+, 561.1485. Found: [M + Na]+,
112.1, 111.9, 53.0, 34.8, 18.3, 13.7. HRMS (ESI): calcd for 561.1480.
C21H14N2O10Na [M + Na]+, 519.1016. Found: [M + Na]+,
519.1011.
N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-2-methyl-
1-oxopropan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-3-carboxa-
(R)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-1-oxo- mide (41). Yellow solid. Mp: 273–276 °C. HPLC: 95.24%, tR
=
hexan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-3-carboxamide (37). 2.228 min. 1H NMR (300 MHz, DMSO-d6) δ 10.70 (br s, 1H),
Yellow solid. Mp: 252–255 °C. HPLC: 96.79%, tR = 3.660 min. 9.97 (br s, 1H), 9.61 (br s, 1H), 9.33 (br s, 1H), 9.12 (s, 1H), 9.00
1H NMR (300 MHz, DMSO-d6) δ 10.66 (br s, 1H), 10.30–9.25 (s, 1H), 8.71 (s, 1H), 8.34 (s, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.01
(br s, 3H), 9.89 (s, 1H), 9.11 (d, J = 7.8 Hz, 1H), 8.77 (s, 1H), (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.7 Hz, 1H), 6.82 (d, J = 8.1 Hz,
8.47 (s, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 1H), 1.60 (s, 6H). 13C NMR (75 MHz, DMSO-d6) δ 174.0, 162.4,
6.91 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 4.99–4.93 161.7, 158.9, 152.9, 149.5, 148.7, 144.7, 140.8, 132.6, 132.5,
(m, 1H), 1.90–1.65 (m, 2H), 1.33 (s, 4H), 0.86 (t, J = 6.7 Hz, 3H). 128.5, 122.3, 120.7, 118.9, 114.3, 113.8, 113.7, 112.7, 112.4,
13C NMR (75 MHz, DMSO-d6) δ 171.5, 161.3, 161.1, 157.7, 57.8, 25.3. HRMS (ESI): calcd for C23H18N2O10Na [M + Na]+,
152.3, 149.0, 148.1, 144.2, 140.3, 132.0, 131.9, 127.7, 121.5, 505.0859. Found: [M + Na]+, 505.0855.
120.2, 118.1, 113.6, 113.1, 112.8, 112.1, 111.8, 53.2, 32.4, 27.1,
N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)carbamoyl)cyclo-
(42).
Yellow solid. Mp: 170–172 °C. HPLC: 95.13%, tR = 3.883 min.
21.9, 13.8. HRMS (ESI): calcd for C25H22N2O10Na [M + Na]+, hexyl)-7,8-dihydroxy-2-oxo-2H-chromene-3-carboxamide
533.1172. Found: [M + Na]+, 533.1166.
(R)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)- 1H NMR (300 MHz, DMSO-d6) δ 10.73 (br s, 1H), 9.94 (br s,
4-methyl-1-oxopentan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene- 1H), 9.61 (br s, 1H), 9.31 (br s, 1H), 9.09 (s, 1H), 9.03 (s, 1H),
3-carboxamide (38). Yellow solid. Mp: 174–178 °C. HPLC: 8.72 (s, 1H), 8.38 (s, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.01 (d, J =
1
98.99%, tR = 3.519 min. H NMR (300 MHz, DMSO-d6) δ 10.65 9.0 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 8.1 Hz, 1H),
(s, 1H), 9.90 (s, 2H), 9.58 (br s, 1H), 9.30 (br s, 1H), 9.04 (d, J = 2.28–2.18 (m, 2H), 1.88–1.74 (m, 2H), 1.72–1.56 (m, 3H),
7.8 Hz, 1H), 8.77 (s, 1H), 8.46 (s, 1H), 7.33 (d, J = 8.4 Hz, 1H), 1.54–1.36 (m, 2H), 1.35–1.20 (m, 1H). 13C NMR (75 MHz,
7.00 (d, J = 8.7 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.81 (d, J = DMSO-d6) δ 173.0, 161.5, 161.4, 158.0, 152.4, 148.9, 148.0,
8.4 Hz, 1H), 5.04–4.94 (m, 1H), 1.74–1.60 (m, 3H), 0.94 (d, J = 144.2, 140.0, 132.1, 131.9, 126.4, 121.6, 120.3, 118.0, 113.8,
5.1 Hz, 6H). 13C NMR (75 MHz, DMSO-d6) δ 171.8, 161.4, 113.2 (two carbons), 112.1, 111.8, 59.9, 31.28 (two carbons),
161.0, 157.7, 152.3, 149.1, 148.1, 144.2, 140.3, 132.0, 131.9, 24.71, 20.92 (two carbons). HRMS (ESI): calcd for
127.7, 121.6, 120.2, 118.1, 113.6, 113.0, 112.7, 112.1, 111.8, C26H22N2O10Na [M + Na]+, 545.1172. Found: [M + Na]+,
52.0, 41.4, 24.5, 23.1, 21.8. HRMS (ESI): calcd for 545.1166.
C25H22N2O10Na [M + Na]+, 533.1172. Found: [M + Na]+,
533.1165.
(S)-N-(1-Cyclohexyl-2-((7,8-dihydroxy-2-oxo-2H-chromen-3-yl)-
amino)-2-oxoethyl)-7,8-dihydroxy-2-oxo-2H-chromene-3-carbox-
N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-1-oxohep- amide (43). Yellow solid. Mp: 190–203 °C. HPLC: 97.83%, tR
=
1
tan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-3-carboxamide (39). 2.992 min. H NMR (300 MHz, DMSO-d6) δ 9.91 (s, 1H), 9.13
1
Grey solid. Mp: 266–267 °C. HPLC: 98.06%, tR = 5.512 min. H (d, J = 8.4 Hz, 1H), 8.77 (s, 1H), 8.47 (s, 1H), 7.33 (d, J = 8.4 Hz,
NMR (300 MHz, DMSO-d6) δ 10.64 (br s, 1H), 9.93 (br s, 1H), 1H), 7.00 (d, J = 8.7 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.80 (d, J =
9.87 (s, 1H), 9.59 (br s, 1H), 9.30 (br s, 1H), 9.11 (d, J = 7.8 Hz, 8.4 Hz, 1H), 5.02–4.85 (m, 1H), 1.89–1.55 (m, 6H), 1.26–1.00
1H), 8.77 (s, 1H), 8.47 (s, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.00 (d, (m, 5H). 13C NMR (75 MHz, DMSO-d6) δ 170.8, 161.4, 161.2,
J = 8.4 Hz, 1H), 6.91 (d, J = 8.7 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 157.7, 152.4, 149.1, 148.1, 144.2, 140.3, 132.0, 131.9, 127.8,
4.99–4.90 (m, 1H), 1.90–1.65 (m, 2H), 1.40–1.20 (m, 6H), 0.85 121.6, 120.1, 118.1, 113.6, 113.0, 112.7, 112.1, 111.8, 57.3, 41.0,
(s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 172.6, 163.1, 162.2, 29.2, 27.8, 25.7, 25.6 (two carbons). HRMS (ESI): calcd for
159.0, 153.2, 150.3, 149.0, 145.0, 141.2, 132.8, 132.7, 129.8, C27H24N2O10Na [M + Na]+, 559.1329. Found: [M + Na]+,
122.9, 120.8, 119.4, 114.7, 114.1, 113.3, 113.0, 112.7, 54.6, 33.0, 559.1325.
31.6, 25.3, 22.7, 14.6. HRMS (ESI): calcd for C26H24N2O10Na
[M + Na]+, 547.1329. Found: [M + Na]+, 547.1323.
(S)-N-(3-Cyclohexyl-1-((7,8-dihydroxy-2-oxo-2H-chromen-3-yl)-
amino)-1-oxopropan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-
N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-1-oxo- 3-carboxamide (44). Yellow solid. Mp: 151–154 °C. HPLC:
1
octan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-3-carboxamide (40). 97.17%, tR = 7.583 min. H NMR (300 MHz, DMSO-d6) δ 9.89
Grey solid. Mp: 252–254 °C. HPLC: 98.68%, tR = 7.652 min. (s, 1H), 9.05 (d, J = 7.5 Hz, 1H), 8.77 (s, 1H), 8.45 (s, 1H), 7.33
1H NMR (300 MHz, DMSO-d6) δ 10.64 (s, 1H), 9.94 (s, 1H), 9.88 (s, (d, J = 8.4 Hz, 1H), 7.00 (d, J = 9.0 Hz, 1H), 6.91 (d, J = 8.4 Hz,
1H), 9.59 (s, 1H), 9.32 (s, 1H), 9.11 (d, J = 8.1 Hz, 1H), 8.77 (s, 1H), 6.81 (d, J = 8.4 Hz, 1H), 5.05–4.95 (m, 1H), 1.85–1.55
1H), 8.47 (s, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.00 (d, J = 8.1 Hz, (m, 6H), 1.45–1.35 (m, 1H), 1.24–1.04 (m, 4H), 1.02–0.84 (m, 2H).
1H), 6.91 (d, J = 7.8 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 4.98–4.90 13C NMR (75 MHz, DMSO-d6) δ 171.9, 161.4, 161.1, 157.7,
(m, 1H), 1.90–1.65 (m, 2H), 1.40–1.15 (m, 8H), 0.84 (t, J = 6.6 152.3, 149.1, 148.1, 144.2, 140.3, 132.0, 131.9, 127.7, 121.6,
Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 171.5, 161.3, 161.1, 120.2, 118.1, 113.6, 113.0, 112.7, 112.1, 111.8, 51.5, 33.8, 33.2,
157.7, 152.3, 149.0, 148.1, 144.2, 140.3, 132.0, 131.9, 127.6, 32.0, 25.9, 25.7, 25.6. HRMS (ESI): calcd for C28H26N2O10Na
121.6, 120.1, 118.1, 113.6, 113.0, 112.8, 112.1, 111.8, 53.2, 32.7, [M + Na]+, 573.1485. Found: [M + Na]+, 573.1476.
3730 | Org. Biomol. Chem., 2014, 12, 3721–3734
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(S)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-1-oxo- 1H), 8.48 (s, 1H), 7.27–7.05 (m, 16H), 6.89 (d, J = 8.4 Hz, 1H),
3-phenylpropan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-3-car- 6.88 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H), 5.13 (t, J =
boxamide (45). Orange solid. Mp: 225–228 °C. HPLC: 95.21%, 6.3 Hz, 1H), 3.08–3.00 (m, 2H).
tR = 3.219 min. 1H NMR (300 MHz, DMSO-d6) δ 10.65 (br s,
(S)-N1-(7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)-2-(7,8-dihydroxy-
1H), 9.99 (s, 1H), 9.09 (d, J = 7.8 Hz, 1H), 8.71 (s, 1H), 8.46 (s, 2-oxo-2H-chromene-3-carboxamido)succinamide (49). To a solu-
1H), 7.35–7.15 (m, 6H), 6.99 (d, J = 8.4 Hz, 1H), 6.90 (d, J = tion of compound 49a (46 mg, 0.0704 mmol) in 3 mL DCM
8.7 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 5.25–5.16 (m, 1H), 3.22 was added 2 mL TFA. The mixture was stirred at rt for 2 h and
(dd, J = 13.8, 4.8 Hz, 1H), 3.03 (dd, J = 13.5, 8.7 Hz, 1H). 13C the yellow solid was isolated by filtration to afford 49 (24 mg,
NMR (75 MHz, DMSO-d6) δ 170.6, 161.3, 160.9, 157.7, 152.4, 77%). Mp: 273–274 °C. HPLC: 95.92%, tR = 2.004 min. 1H
149.1, 148.1, 144.2, 140.2, 136.8, 132.1, 131.9, 129.3 (two NMR (300 MHz, DMSO-d6) δ 10.67 (br s, 1H), 9.92 (br s, 1H),
carbons), 128.1 (two carbons), 127.3, 126.5, 121.6, 120.2, 118.1, 9.64 (s, 2H), 9.43 (d, J = 7.5 Hz, 1H), 9.32 (br s, 1H), 8.80 (s,
113.7, 113.1, 112.4, 112.1, 111.8, 54.6, 38.0. HRMS (ESI): calcd 1H), 8.48 (s, 1H), 7.52 (s, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.04 (s,
for C28H20N2O10Na [M + Na]+, 567.1016. Found: [M + Na]+, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.1 Hz, 1H), 6.81 (d, J =
567.1005.
8.7 Hz, 1H), 5.08–4.99 (m, 1H), 2.73 (d, J = 5.7 Hz, 2H). 13C
(S)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)- NMR (75 MHz, DMSO-d6) δ 171.6, 170.1, 161.7, 160.8, 157.7,
4-(methylthio)-1-oxobutan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene- 152.4, 149.2, 148.0, 144.3, 140.0, 132.1, 131.9, 126.0, 121.7,
3-carboxamide (46). Yellow solid. Mp: 174–176 °C. HPLC: 120.3, 118.0, 113.6, 113.1, 112.5, 112.1, 111.8, 50.6, 37.0.
1
95.60%, tR = 2.580 min. H NMR (300 MHz, DMSO-d6) δ 10.60 HRMS (ESI): calcd for C23H17N3O11Na [M + Na]+, 534.0761.
(br s, 1H), 10.30–9.25 (br s, 3H), 9.88 (s, 1H), 9.18 (d, J = 7.8 Found: [M + Na]+, 534.0765.
Hz, 1H), 8.76 (s, 1H), 8.47 (s, 1H), 7.34 (d, J = 9.0 Hz, 1H), 7.01
(S)-tert-Butyl (5-((7,8-bis(methoxymethoxy)-2-oxo-2H-chromen-
(d, J = 9.0 Hz, 1H), 6.91 (d, J = 8.7 Hz, 1H), 6.81 (d, J = 8.1 Hz, 3-yl)amino)-4-(7,8-bis(methoxymethoxy)-2-oxo-2H-chromene-
1H), 5.05–4.95 (m, 1H), 2.58–2.50 (m, 2H), 2.21–2.10 (m, 1H), 3-carboxamido)-5-oxopentyl)carbamate (50a). 1H NMR
2.06 (s, 3H), 2.04–1.93 (m, 1H). 13C NMR (75 MHz, DMSO-d6) (300 MHz, CDCl3) δ 9.24 (d, J = 7.2 Hz, 1H), 8.91 (s, 1H), 8.87
δ 170.7, 161.6, 161.0, 157.7, 152.3, 149.0, 148.2, 144.2, 140.3, (s, 1H), 8.63 (s, 1H), 7.40 (d, J = 8.7 Hz, 1H), 7.21 (d, J = 8.7 Hz,
132.1, 131.9, 127.8, 121.6, 120.1, 118.1, 113.6, 113.1, 112.8, 1H), 7.16 (d, J = 8.7 Hz, 1H), 7.11 (d, J = 8.7 Hz, 1H), 5.32
112.1, 111.8, 52.8, 32.6, 29.3, 14.7. HRMS (ESI): calcd for (s, 2H), 5.25 (s, 4H), 5.22 (s, 2H), 4.85–4.75 (m, 1H), 4.65 (br s,
C24H20N2O10SNa [M + Na]+, 551.0736. Found: [M + Na]+, 1H), 3.71 (s, 3H), 3.67 (s, 3H), 3.52 (s, 3H), 3.50 (s, 3H),
551.0732.
3.28–3.13 (m, 2H), 2.20–2.05 (m, 1H), 1.97–1.82 (m, 1H), 1.68
(S)-tert-Butyl 4-((7,8-dihydroxy-2-oxo-2H-chromen-3-yl)amino)- (d, J = 7.2 Hz, 2H), 1.43 (s, 9H).
3-(7,8-dihydroxy-2-oxo-2H-chromene-3-carboxamido)-4-oxo-
(S)-N-(5-Amino-1-((7,8-dihydroxy-2-oxo-2H-chromen-3-yl)-
butanoate (47). Yellow solid. Mp: 255–258 °C. HPLC: 95.80%, amino)-1-oxopentan-2-yl)-7,8-dihydroxy-2-oxo-2H-chromene-
1
tR = 3.192 min. H NMR (300 MHz, DMSO-d6) δ 9.63 (s, 1H), 3-carboxamide (50). To a solution of compound 50a (28 mg,
9.38 (d, J = 7.5 Hz, 1H), 8.81 (s, 1H), 8.46 (s, 1H), 7.36 (d, J = 0.0458 mmol) in 2 mL DCM was added 2 mL of 2 M HCl in
8.4 Hz, 1H), 7.01 (d, J = 8.1 Hz, 1H), 6.91 (d, J = 8.7 Hz, 1H), EtOAc. The mixture was stirred at rt for 2 h and the solvent
6.81 (d, J = 8.9 Hz, 1H), 5.15–5.06 (m, 2H), 2.84 (d, J = 5.7 Hz, was evaporated to afford 50 as a yellow solid (18 mg, 72%).
2H), 1.37 (s, 9H). 13C NMR (75 MHz, DMSO-d6) δ 169.6, 169.2, Mp: 233–235 °C. HPLC: 95.81%, tR = 1.520 min. 1H NMR
161.8, 160.9, 157.7, 152.6, 149.4, 148.2, 144.3, 140.2, 132.1, (300 MHz, CD3OD) δ 9.54 (d, J = 7.5 Hz, 1H), 8.78 (s, 1H),
131.9, 126.8, 121.8, 120.2, 118.2, 113.7, 113.2, 112.4, 112.1, 8.51 (s, 1H), 7.23 (d, J = 9.0 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H),
111.8, 80.7, 50.5, 37.5, 27.6. HRMS (ESI): calcd for 6.92 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 4.96–4.89
C27H24N2O10Na [M + Na]+, 591.1227. Found: [M + Na]+, 591.1222. (m, 1H), 3.03 (t, J = 6.9 Hz, 2H), 2.18–2.06 (m, 1H), 2.04–1.91
(S)-4-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-3-(7, (m, 1H), 1.90–1.78 (m, 2H). 13C NMR (75 MHz, CD3OD)
8-dihydroxy-2-oxo-2H-chromene-3-carboxamido)-4-oxobutanoic δ 172.4, 164.8, 163.2, 160.0, 154.1, 151.1, 149.8, 145.8,
acid (48). Compound 47 (40 mg, 0.0704 mmol) was dissolved 141.8, 133.6, 133.5, 129.6, 123.1, 121.5, 119.8, 115.0,
in 4 mL formic acid and stirred at rt for 6 h. The solvent was 114.4, 113.9, 113.7, 113.6, 54.9, 40.6, 30.7, 25.0. HRMS (ESI):
evaporated and the residue was purified by column chromato- calcd for C24H22N3O10 [M + H]+, 512.1305. Found: [M + H]+,
graphy (DCM–MeOH–AcOH = 10 : 1 : 1) to give 48 as a yellow 512.1293.
solid (13 mg, 36%). Mp: >280 °C. HPLC: 95.57%, tR
2.364 min. H NMR (300 MHz, DMSO-d6) δ 9.70 (s, 1H), 9.40 4-methyl-1-oxopentan-2-yl)-7,8-dimethoxy-2-oxo-2H-chromene-
(d, J = 7.5 Hz, 1H), 8.72 (s, 1H), 8.47 (s, 1H), 7.30 (d, J = 8.4 Hz, 3-carboxamide (51). Pale solid. Mp: 199–200 °C. HPLC:
=
(R)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-
1
1
1H), 7.01 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 2H), 5.08–5.02 95.70%, tR = 6.199 min. H NMR (300 MHz, DMSO-d6) δ 9.95
(m, 1H), 2.84 (d, J = 6.0 Hz, 2H). HRMS (ESI): calcd for (s, 1H), 9.93 (s, 1H), 9.32 (s, 1H), 9.01 (d, J = 8.1 Hz, 1H), 8.84
C23H16N2O12Na [M + Na]+, 535.0601. Found: [M + Na]+, (s, 1H), 8.46 (s, 1H), 7.75 (d, J = 9.3 Hz, 1H), 7.24 (d, J = 8.7 Hz,
535.0598.
1H), 7.00 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 5.05–4.96
(S)-N1-(7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)-2-(7,8-dihydroxy- (m, 1H), 3.96 (s, 3H), 3.86 (s, 3H), 1.75–1.60 (s, 3H), 0.94 (d, J =
2-oxo-2H-chromene-3-carboxamido)-N4-tritylsuccinamide (49a). 4.8 Hz, 6H). 13C NMR (75 MHz, DMSO) δ 171.8, 161.1, 160.6,
White solid. 1H NMR (300 MHz, CD3OD + CDCl3) δ 8.72 (s, 157.7, 157.2, 148.5, 148.1, 147.8, 140.3, 134.8, 132.0, 127.8,
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126.1, 120.2, 118.1, 114.6, 113.1, 113.0, 112.1, 110.3, 60.8, 56.6, ELISA kinase assay
52.0, 41.3, 24.5, 23.2, 21.8. HRMS (ESI): calcd for
Met tyrosine kinase activity was evaluated according to the fol-
lowing procedure: briefly, in enzyme-linked-immunosorbent
assay (ELISA), 20 μg mL−1 poly (Glu,Tyr)4:1 (Sigma) was pre-
coated as a substrate in 96-well plates. 50 μL of 10 μmol L−1
ATP solution diluted in kinase reaction buffer (50 mmol L−1
HEPES pH 7.4, 50 mmol L−1 MgCl2, 0.5 mmol L−1 MnCl2,
0.2 mmol L−1 Na3VO4, 1 mmol L−1 DTT) was added to each
well. 1 μL of various concentrations of indicated compounds
diluted in 1% DMSO (v/v) (Sigma) was added to each reaction
well. 1% DMSO (v/v) was used as a negative control. The
kinase reaction initiated after the addition of purified tyrosine
kinase proteins diluted in 49 μL of kinase reaction buffer solu-
tion. After incubation for 60 min at 37 °C, the plate was
washed three times with phosphate buffered saline (PBS) con-
taining 0.1% Tween 20 (T-PBS). 100 μL anti-phosphotyrosine
(PY99) antibody (1 : 500 diluted in 5 mg mL−1 BSA T-PBS) was
then added. After 30 min incubation at 37 °C, the plate was
washed three times. 100 μL horseradish peroxidase-conjugated
goat anti-mouse IgG (1 : 2000 diluted in 5 mg mL−1 BSA T-PBS)
was added. The plate was then incubated at 37 °C for 30 min,
and washed 3 times. 100 μL of a solution containing 0.03%
H2O2 and 2 mg ml−1 o-phenylenediamine in 0.1 mol L−1
citrate buffer, pH 5.5, was added. The reaction was terminated
by the addition of 50 μL of 2 mol L−1 H2SO4 as the color
changed, and the plate was read using a multi-well spectro-
photometer (SpectraMAX 190, Molecular Devices) at 490 nm.
The inhibition rate (%) was calculated using the following
equation: [1 − (A490/A490 control)] × 100%. IC50 values were cal-
culated from the inhibition curves from two separate exper-
iments. For ATP competition assay, various concentrations of
ATP were diluted for the kinase reaction.
C27H26N2O10Na [M + Na]+, 561.1485. Found: [M + Na]+,
561.1476.
(R)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-
4-methyl-1-oxopentan-2-yl)-7-hydroxy-2-oxo-2H-chromene-
3-carboxamide (52). Pale solid. Mp: 192–193 °C. HPLC:
1
97.67%, tR = 5.108 min. H NMR (300 MHz, DMSO-d6) δ 11.14
(s, 1H), 10.00 (s, 1H), 9.90 (s, 1H), 9.33 (s, 1H), 9.01 (d, J = 7.8
Hz, 1H), 8.80 (s, 1H), 8.44 (s, 1H), 7.82 (d, J = 8.4 Hz, 1H), 6.99
(d, J = 8.4 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.82–6.79 (m, 2H),
5.02–4.92 (m, 1H), 1.75–1.62 (m, 3H), 0.93 (d, J = 4.8 Hz, 6H).
13C NMR (75 MHz, DMSO-d6) δ 171.9, 163.9, 161.4, 161.2,
157.8, 156.4, 148.6, 148.2, 140.3, 132.2, 132.1, 127.9, 120.2,
118.2, 114.5, 113.1 (two carbons), 112.1, 111.2, 101.9, 52.0,
41.4, 24.6, 23.2, 21.8. HRMS (ESI): calcd for C25H22N2O9Na
[M + Na]+, 517.1223. Found: [M + Na]+, 517.1215.
(R)-N-(1-((7,8-Dihydroxy-2-oxo-2H-chromen-3-yl)amino)-
4-methyl-1-oxopentan-2-yl)-8-hydroxy-2-oxo-2H-chromene-3-car-
boxamide (53). Pale solid. Mp: 179–182 °C. HPLC: 97.89%, tR
= 4.291 min. 1H NMR (300 MHz, DMSO-d6) δ 10.45 (s, 1H),
9.95 (s, 2H), 9.33 (s, 1H), 9.09 (d, J = 7.8 Hz, 1H), 8.84 (s, 1H),
8.46 (s, 1H), 7.43–7.36 (m, 1H), 7.25–7.21 (m, 2H), 7.00 (d, J =
8.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 5.06–4.96 (m, 1H),
1.73–1.62 (m, 3H), 0.95 (d, J = 4.2 Hz, 6H). 13C NMR (75 MHz,
DMSO-d6) δ 171.7, 160.9, 160.4, 157.7, 148.4, 148.2, 144.4,
142.6, 140.3, 132.0, 127.8, 125.1, 120.3, 120.2 (two carbons),
118.1, 113.0, 112.1, 52.0, 41.4, 24.52, 23.1, 21.8. HRMS (ESI):
calcd for C25H22N2O9Na [M
[M + Na]+, 517.1217.
+
Na]+, 517.1223. Found:
(R)-7-Hydroxy-N-(1-((7-hydroxy-2-oxo-2H-chromen-3-yl)amino)-
4-methyl-1-oxopentan-2-yl)-2-oxo-2H-chromene-3-carboxamide
(54). Beige solid. Mp: 175–176 °C. HPLC: 96.22%, tR
=
6.802 min. 1H NMR (300 MHz, DMSO-d6) δ 11.10 (s, 1H), 10.39 Western blot analysis
(s, 1H), 9.92 (s, 1H), 9.01 (d, J = 7.9 Hz, 1H), 8.81 (s, 1H), 8.50
EBC-1 and BaF3/TPR-Met cells were treated with indicated
(s, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 6.89
(dd, J = 8.4 Hz, 1.2 Hz, 1H), 6.85–6.70 (m, 3H), 5.03–4.98 (m,
1H), 1.68–1.66 (m, 3H), 0.94 (d, J = 5.1 Hz, 6H). 13C NMR
(75 MHz, DMSO-d6) δ 171.8, 163.8, 161.3, 161.1, 159.8, 157.8,
156.4, 151.7, 148.5, 132.1, 129.1, 127.2, 120.4, 114.4, 113.6,
113.0, 111.2, 111.1, 101.9, 101.8, 52.0, 41.4, 24.5, 23.1, 21.8.
HRMS (ESI): calcd for C25H22N2O8Na [M + Na]+, 501.1274.
Found: [M + Na]+, 501.1264.
compounds for 4 h at 37 °C and then lysed in 1× SDS sample
buffer. The cell lysates were subsequently resolved on 10%
SDS-PAGE and transferred to nitrocellulose membranes. Mem-
branes were probed with appropriate primary antibodies
(c-Met [Santa Cruz] and phospho-c-Met [Cell Signaling Tech-
nology], and GAPDH [KangChen Biotech] antibody), and then
subsequently with horseradish peroxidase-conjugated anti-
rabbit or anti-mouse IgG. Immunoreactive proteins were
detected using enhanced chemiluminescence detection
reagent (Thermo Fisher).
(R)-8-Hydroxy-N-(1-((7-hydroxy-2-oxo-2H-chromen-3-yl)amino)-
4-methyl-1-oxopentan-2-yl)-2-oxo-2H-chromene-3-carboxamide
(55). Pale solid. Mp: 158–160 °C. HPLC: 98.90%, tR
=
5.604 min. 1H NMR (300 MHz, DMSO-d6) δ 10.45 (s, 1H), 10.38
(s, 1H), 9.96 (s, 1H), 9.09 (d, J = 8.1 Hz, 1H), 8.84 (s, 1H), 8.50
(s, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.44–7.35 (m, 1H), 7.26–7.22
Abbreviations
(m, 2H), 6.79 (d, J = 8.4 Hz, 1H), 6.74 (s, 1H), 5.06–4.96 (m, NSCLC Non-small cell lung cancer
1H), 1.74–1.62 (m, 3H), 0.94 (d, J = 4.5 Hz, 6H). 13C NMR EGFR
(75 MHz, DMSO-d6) δ 171.7, 160.9, 160.4, 159.8, 157.8, 151.8, PKA
148.4, 144.4, 142.6, 129.1, 127.3, 125.1, 120.4, 120.3, 120.1, PKC
119.3, 118.1, 113.6, 111.2, 101.9, 52.0, 41.4, 24.5, 23.1, 21.8. SAR
HRMS (ESI): calcd for C25H22N2O8Na [M + Na]+, 501.1274. Leu
Epidermal growth factor receptor
Protein kinase A
Protein kinase C
Structure–activity relationship
Leucine
N,N-Dimethylformamide
Found: [M + Na]+, 501.1270.
DMF
3732 | Org. Biomol. Chem., 2014, 12, 3721–3734
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Paper
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DCM
Dichloromethane
DMAP 4-Dimethylaminopyridine
BOC
t-Butyloxycarbonyl
12 K. Matsumoto, T. Nakamura, K. Sakai and T. Nakamura,
Proteomics, 2008, 8, 3360–3370.
EDCI
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride
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Q. Chen, S.-Y. Ho, L. Li, S. Kaufman, K. McDorman,
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MOM
Fluorenylmethoxycarbonyl
Methoxymethyl
Acknowledgements
This work was supported by grants from the National Natural
Science Foundation of China (30725049, 81321092, 81102461,
91229205), the National Science and Technology Major Pro-
jects for Major New Drugs Innovation and Development
(2012ZX09304011), the National Program on Key Basic
Research Project of China (no. 2012CB910704), and the
National S&T Major Projects (2012ZX09301001-007).
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