A new family of densely functionalized fused-benzoquinones as potent human protein kinase CK2 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.12.058

A new series of 2-amino-4-phenyl-6-hydroxy-7-alkyl-pyranobenzoquinones was synthesized as ATP-competitive CK2 inhibitors. They were readily synthesized through a three-component Knoevenagel condensation-Michael addition-heterocyclization reaction from ald

Full text of DOI:10.1016/j.ejmech.2017.12.058

Accepted Manuscript
A new family of densely functionalized fused-benzoquinones as potent human protein
kinase CK2 inhibitors
Pedro Martín-Acosta, Samer Haider, Ángel Amesty, Dagmar Aichele, Joachim Jose,
Ana Estévez-Braun
PII:
S0223-5234(17)31089-9
10.1016/j.ejmech.2017.12.058
EJMECH 10036
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 October 2017
Revised Date: 14 December 2017
Accepted Date: 15 December 2017
Please cite this article as: P. Martín-Acosta, S. Haider, Á. Amesty, D. Aichele, J. Jose, A. Estévez-
Braun, A new family of densely functionalized fused-benzoquinones as potent human protein kinase
CK2 inhibitors, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.058.
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ACCEPTED MANUSCRIPT
A new family of densely functionalized fused-benzoquinones as potent
human protein kinase CK2 inhibitors
Pedro Martín-Acosta,a Samer Haider,b Ángel Amesty,a Dagmar Aichele,b Joachim
Jose,b Ana Estévez-Brauna,*
aInstituto Universitario de Bio-Orgánica Antonio González (CIBICAN), Departamento
de Química Orgánica, Universidad de La Laguna, Avda. Astrofísico Francisco Sánchez
Nº 2, 38206, La Laguna, Tenerife, Spain
bInstitute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westfälische
Wilhelms-Universität Münster, Corrensstrasse 48, 48149 Münster, Germany
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Abstract
A new series of 2-amino-4-phenyl-6-hydroxy-7-alkyl-pyranobenzoquinones was
synthesized as ATP-competitive CK2 inhibitors. They were readily synthesized through
a three-component Knoevenagel condensation-Michael addition-heterocyclization
reaction from aldehydes, malononitrile, and 3-alkyl-2,5-dihydroxybenzoquinones. Some
of the synthesized compounds presented interesting inhibitory activity with IC50 values
in the submicromolar range. A structure-activity relationship study was carried out and
the mode of binding was analysed by docking studies and supported by ATP
competition assays.
CK2 INHIBITORS
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1. Introduction
Protein kinase CK2 is a highly conserved serine/threonine protein kinase that is
ubiquitously distributed in multiple cell types [1]. It is a holoenzyme comprises a
heterotetramer consisting of two catalytic (α and/or α’) and two regulatory (β) subunits.
It is recognized as one of the most pleiotropic protein kinase with more than 300
substrates [2], many of which have been confirmed to play crucial roles in the
dysfunction of programmed cell apoptosis and death, migration, differentiation and
oncogenic transformation [3-6]. Overexpression of CK2 has been observed in many
cancers, including hematologic cancers such as acute myelogenous leukemia (AML),
chronic lymphocytic leukemia (CLL), T-cell acute lymphoblastic leukemia (T-ALL)
and multiple myeloma (MM) [7-10]. Downregulation of CK2, either by transfection of
specific siRNA or plasmid-based expression of kinase-inactive CK2, resulted in
reduction of cancer cell viability and induction of apoptosis [11-13]. Thus, CK2 is
becoming an important target for the treatment of cancer and other life-threatening
diseases. Several compounds, belonging to different classes of chemical compounds
like anthraquinones, xanthenones, coumarins, pyrazolo-triazynes, peptides,
benzimidazoles and polyoxygenated benzotriazoles have been reported as CK2
inhibitors [14-23]. However until now, only one of them, the pyridoquinoline CX-4945,
has entered in Phase II clinical trials as potencial anticancer drug [24-27].
Herein, we report the structural optimization and docking studies of a new
family of ATP-competitive CK2 inhibitors with the 2-amino-3-cyano-4H-
pyranobenzoquinone core.
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2. Results and discussion
Most of the CK2 inhibitors known today are small and flat scaffolds having
several rings, which fits into the active site of the CK2 and competes with ATP [14].
One of the representative CK2 inhibitors is the anthraquinone emodin [28]. Due to our
interest in antitumoral quinones [29-33], a set of 60 structurally diverse natural and
synthetic quinones [31] was screened on CK2 activity. From this screening compound 1
emerged with an IC50 < 1 µM.
Compound 1 was obtained through a multicomponent reaction from the natural
benzoquinone embelin (I) [34], 4-chlorobenzaldehyde (II) and malonotrile (III). Based
on calculated values of the Fukui function (f-) as local density functional descriptor
[32], the synthesis of this compound can be rationalized by the initial formation of a
conjugated electron-deficient enone (A) through a Knoevenagel condensation of
embeline (I) and 4-chlorobenzaldehyde (III). The next step of this mechanism could
involve a Michael addition of malononitrile (II) to the reactive quinone methide
intermediate to yield the intermediate (B), which experiments an intramolecular
cyclization to yield the intermediate C that tautomerizes to compound 1 (Scheme 1).
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Scheme 1. Structure and Plausible Formation of Compound 1.
Compound 1 was tested for its inhibitory activity towards the human CK2
holoenzyme following the procedure described earlier [22, 35]. The synthetic peptide
RRRDDDSDDD was used as a substrate. For initial testing, inhibition was determined
at inhibitor concentrations of 10 µM in DMSO as solvent. IC50 value was determined by
measuring CK2 inhibition at eight different concentrations ranging from 0.001 to 100
µM in appropriate intervals and calculated from the resulting dose-response curve [35].
Compound 1 was active toward the target enzyme with an IC50 value of 0.90 ± 0.05
µM. As a control the well-known CK2 inhibitor, emodin was subjected to the same test
conditions which had an IC50 value of 0.58 ± 0.05 µM (Fig. 1).
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100
100
50
0
50
Emodin
Compound
1
0
10- 3
10- 2
10- 1
100
101
102
10-3
10-2
10- 1
100
101
102
Concentration [µM]
Concentration [µM]
Fig. 1. IC50 value determination of compound 1 and emodin with human protein
kinase CK2.
The binding pattern of 1 was analyzed by flexible molecular docking. The
compound inserted into the narrow ATP binding site of CK2 (Fig. 2). As shown the
aliphatic chain of 1 was located at the edge of the pocket and established van der Waals
interactions with the side chains residues of Ile95, Met163, Val116, Val66 and Ile174.
Two hydrogen bonds were observed between the hydroxyl group of 1 and Lys 68, and
between the cyano group and Arg47. Other representative interaction was a π-π
interaction between the aromatic ring and His160. Based on the binding mode, the
presence of different substituents at the phenyl ring to enhance the contact with CK2,
modifications in the length side chain to locate the ligand more deeply within the ATP
binding site, and the replacement of the cyano moiety by other electron withdrawing
groups may increase the inhibitory activity.
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Fig. 2. Binding mode prediction of 1 with CK2 (PDB 3PE1).
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Thus, the SAR of this class of compounds was investigated by synthesizing structural
variations at each of the three sites designated as SAR 1, SAR 2 and SAR 3.
Scheme 2. Modular modifications to SAR1, SAR2 and SAR3 compounds.
In the SAR 1 study, the synthesis was driven by preserving the original
functionalities for the SAR 2 and SAR 3 regions in the lead compound, while the
SAR 1 position was varied. Various functional groups, such as substituted phenyls,
heterocycles and alkyls were introduced in SAR 1. Table 1 shows the structures of
SAR 1 compounds and CK2 inhibitory activity.
Table 1. Structure of SAR 1 compounds and CK2 inhibitory activity
IC50 + SD (µM)b
Compound
R1
Inhibition (%)a
1
-Ph-4-Cl
81
0.90 + 0.052
2
-Ph-4-Br
48
ndc
3
4
-Ph-4-F
-Ph-3-F
50
74
nd
0.996 + 0.06
5
6
7
-Ph-3-F-4-OMe
-Ph-4-CN
49
60
90
nd
nd
-Ph-4-NO2
0.22 + 0.04
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8
9
-Ph-3-NO2
-Ph-4-CF3
81
58
88
0.89 + 0.024
nd
10
-Ph-4-COOH
1.36 + 0.19
11
12
-Ph-4-COOMe
-Ph
64
76
nd
0.57 + 0.038
13
14
15
16
-Ph-4-OMe
-Ph-3,4-(OMe)2
-Ph-3,4-OCH2O-
-3-pyridin
78
38
73
71
0.84 + 0.16
nd
1.94 + 0.16
1.38 + 0.094
17
18
19
20
-4-pyridin
-2-furyl
86
66
37
11
nid
75
1.44 + 0.4
0.72 + 0.016
-cyclohexyl
-C(CH3)3
nd
nd
21
22
-CH2CH3
-(CH2)5CH3
nd
3.71 + 0.079
Emodin
99
0.58 + 0.05
a
The percent inhibition of CK2 activity was determined for each compound at a fixed
concentration of 10 µM.
b
For the best compounds producing at least 65% inhibition at 10 µM, the concentration
was varied to determine the IC50.
c nd: not determined
d ni: no inhibition
The replacement of chlorine in para position by other halogens such as bromine
or fluorine (Table 1, compounds 2-3) led to loss of activity, while the activity was
retained when the fluorine was located at meta-position. The effect of withdrawing
groups like -CN, -NO2, -CF3, -COOH and -COOMe (Table 1, compounds 6-11) was
analysed. The introduction of a nitro group at the para position of the phenyl group (7)
showed 4-fold improvement of the CK2 inhibitory activity compared to the lead
compound 1. Compound 8 having the nitro group at the meta position was as active as
compound 1, and derivative -Ph-4-COOH (10) resulted slightly less active than 1. The
rest of the compounds with electron-withdrawing groups (6, 9, 11) did not show good
activity, with inhibition percentage lower than 65%. Compound 12 with an
unsubstituted phenyl group showed a slightly better activity than compound 1.
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Regarding the analogues with electron-donating groups (13-15), compound 13 (-Ph-4-
OMe) showed slightly better activity than 1, and a loss of activity was observed with
additional electron-donating groups (14-15). Substitutions of the phenyl groups with
heterocyclic rings were also evaluated (compounds 16-18), the best activity for this
series was achieved with the 2-furyl derivative (0.72 ± 0.016) while 3-pyridin and 2-
pyridin analogues resulted 1.4 and 1.6-fold less active than 1. The introduction of non-
aromatic moieties in the SAR1 position (19-22) resulted in loss of CK2 inhibitory
activity.
On the basis of the SAR1 findings, a phenyl group or heteroaromatic ring was
necessary for the activity, which is supported by the existence of a π-π interaction
between the aromatic moiety and His-160 in the Docking model. The different
substituents at para- or meta- position modulate the CK2 inhibitory activity depending
on the effectiveness of the interactions they establish with the corresponding residues.
Thus for instance, the best activity achieved by compound 7 having a 4-NO2 group can
be explained on the basis of four hydrogen bond interactions: between the amino group
and Leu 45, between the cyano group and Arg 47, between the OH and Lys 68, and
between the NO2 group and Lys 158. The aliphatic chain of compound 7 was also
located at the edge of the pocket in the same way as compound 1 and established van
der Waals interactions with amino acid residues such as Ile95, Ile174, Val 116, Val66
and Phe113 (Fig. 3). Finally, the π-π interaction between the aromatic ring and His 160
was also observed. The interactions with His-160 and Lys-68 are similar to those
observed for the potent CK2 inhibitor CX-4945.
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Fig. 3. Binding mode prediction of 7 with CK2 (PDB 3PE1).
Next, our exploration shifted to modifications at the lateral chain of the
benzoquinone nucleus (SAR 2). Considering that the CK2 inhibitory activity is
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favoured by the presence of the -Ph-4-NO2 (SAR 1), the subsequent analogues were
investigated with this moiety retained. The influence of the side chain length on the
CK2 inhibitory was analysed with the preparation of compounds 23 (R2= (CH2)7CH3),
24 (R2= (CH2)5CH3), 25 (R2= (CH2)3CH3) and 26 (R2= CH2CH3).
These compounds were obtained from the multicomponent reaction of 4-
nitrobenzaldehyde, malononitrile and the corresponding benzoquinones IV, V, VI and
VII. The benzoquinones (IV-VII) were synthesized following the reactions shown in
Scheme 3 [36].
Scheme 3. Preparation of benzoquinones (IV-VII).
Table 2 shows the results obtained in the evaluation of derivatives (23-26). As we can
see the shortening of the side chain produces a drastic loss of activity. In docking
model, compound 24 (R2=(CH2)5CH3), for example, showed the same pose and similar
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interactions as compound 7 into the binding pocket except for the interactions
established by the alkyl chain. The inactivity of this compound could be explained for
the absence of the hydrophobic interactions that the C-7-C-11 fragment of the alkyl
chain of compound 7 establish, and reinforces the key role that the C-11 plays driving
the orientation of the compounds within the lining of the ATP-binding site.
Table 2. Structure of SAR 2 compounds and CK2 inhibitory activity.
Compound
R2
Inhibition (%)a
IC50 ± SD (µM)
6
-(CH2)10CH3
-(CH2)7CH3
-(CH2)5CH3
-(CH2)3CH3
-CH2CH3
90
80
nia
26
26
0.22 ± 0.04
23
24
25
26
1.99 ± 0.19
ndb
nd
nd
a ni: no inhibition
b nd: not determined
Attending to the SAR 2 region we also evaluated the derivatives 27 and 28 in
order to see the effect of the replacement of the alkyl chain and the hydroxyl group by a
fused aromatic ring, and also the replacement of the free hydroxyl by a methoxy group.
In both cases low percentages of inhibition were obtained (37% (27), 39% (28)), which
ratifies the importance of the C-11 alkyl chain and the free hydroxyl group for the
activity (Fig. 4).
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Fig. 4. Structures of derivatives 27 and 28.
Next, our attention turned to the SAR 3 series bearing the -Ph-4-NO2 (SAR 1), -
(CH2)10 CH3 and the free hydroxyl (SAR 2) moieties on the structural template.
Since the NH2 group in the docking model establishes a hydrogen bond
interaction with Leu 45, the importance of this group for the activity was assumed, and
the SAR 3 was focused in the replacement of the CN group with electron-withdrawing
groups, which could also act as hydrogen bond acceptors.
Because of the CN group is introduced by the malononitrile component in the MCR, we
carried out the MCR with other methylene active compound bearing one cyano group
(necessary to generate the NH2 functionality) and other electron-withdrawing groups
such as methylformyloxy or ethylformyloxy. Replacement of the cyano moiety by these
groups (29-30) resulted in loss of activity. Compounds 29 and 30 with a
methylformyloxy and an ethylformyloxy groups, respectively, showed a similar pose
respect to compound 7. The carbonyl of the methylformyloxy and ethylformyloxy
moieties presents the same interaction that the cyano group establishes with Arg 47. The
loss of activity of these compounds could be explained considering a plausible
intramolecular hydrogen bond between the NH2 group and the corresponding methoxy
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or ethoxy group, which consequently debilities the interaction between the amino group
and Leu 45.
Table 3. Structure of SAR 3 compounds and CK2 inhibitory activity.
Compound
R3
Inhibition (%)a
IC50 ± SD (µM)
7
-CN
90
58
53
0.22 ± 0.04
29
-COOCH3
nda
nd
30
-COOCH2CH3
a nd: not determine
The effect of the lead compound 1 (IC50: 0.90 µM) on viability of MCF-7 breast
cancer cells was tested using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazoliumbromide) assay and compared to the effect of compounds 4 (IC50: 1 µM), 7
(0.22 µM), 8 (0.89 µM), 12 (0.57 µM), and 13 (0.84 µM). As it is shown in Fig. 5, all
compounds tested showed almost no cytotoxic effect in MCF-7 cells after 48 h
incubation at concentration of 10 µM. While using 100 µM (which is far away from any
therapeutic dosage) the cytotoxicity was between 50-90%. The highest effect was
shown by compound 7 with around 90 % inhibition, while compound 12 showed the
weakest effect with around 50 %, and the inhibition effects of other compounds were
for 4, 8, and 13 around 60%, 65%, and 55%, respectively.
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Fig. 5. Effects of five selected inhibitors (1, 4, 7, 8, 12, and 13) on the cell viability of
MCF-7 cells evaluated by MTT assay, after 48h of incubation. Control cells contained
1% DMSO and the results are presented as mean values (± SD) of three assays.
Columns in dark gray represents % cell viability after incubation with 100 µM of the
compounds. Columns in light gray represents the effect in 10 µM.
In order to validate the ATP competitive mode of inhibition, compound 12 was
selected as a representative inhibitor of this series, and the IC50 values were determined
at eight different concentrations of compound 12 ranging from 0.001 to 100 µM, and
repeated four times using different ATP concentrations each time. The IC50 values
were observed to increase linearly with the ATP concentration, indicating the ATP
competitive mode of CK2 inhibition (Fig. 6). The Ki value of CK2 inhibitory by
compound 12 was determined from the IC50 values obtained at various ATP
concentrations. The regression line showed a Y-axis intercept at an IC50 value of 0.39
µM (Fig. 6) and thereby defined the Ki value of compound 12 to be at this
concentration.
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4
3
2
1
0
0
20
40
60
80
100
120
ATP [µM]
Fig. 6. ATP-competitive inhibition of human CK2 by compound 12. Four IC50 values
with different ATP concentrations were determined using eight different concentration
of the inhibitor ranging from 0.001 to 100 µM and plotted against the respective ATP
concentrations. Each IC50 value was determined 3 times independently. Mean values
with corresponding standard deviation are given. The Ki value is defined as the Y-
intercept and was calculated to be 0.39 µM (R2= 0.93).
3. Conclusion
From the screening on CK2 activity of a set of 60 structurally diverse natural and
synthetic quinones, an embelin derivative (1) with an IC50 = 0.90 ± 0.052 µM was
identified. The SAR of the compound class was investigated by synthesizing structural
variations and by determination of their CK2 inhibitory activities. It was possible to
enhance the activity of compound (1) and an IC50 value of 0.22 ± 0.04 µM was achieved
for the most active compound. The cell viability for some compounds was determined,
and the binding pattern of these compounds into the ATP binding site of CK2 was
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analysed by molecular docking, and it also was corroborated by ATP competitive
assay. This family of inhibitors is a promising start point for further chemical
modifications focused on the nature of the heterocyclic ring fused to the
benzoquinone core to obtain more efficient CK2 inhibitors.
4. Experimental Section
4.1 General Experimental Procedures. IR spectra were obtained using a Fourier
Transform Infrared spectrometer. NMR spectra were recorded in CDCl3 or DMSO at
1
13
500 or 600 MHz for H NMR and 125 or 150 MHz for C NMR. Chemical shifts are
1
given in (δ) parts per million and coupling constants (J) in hertz (Hz). H and 13C
spectra were referenced using the solvent signal as internal standard. Melting points
were taken on a capillary melting point apparatus and are uncorrected. HREIMS were
recorded using a high-resolution magnetic trisector (EBE) mass analyzer. Analytical
thin-layer chromatography plates used were Polygram-Sil G/UV254. Preparative thin-
layer chromatography was carried out with Analtech silica gel GF plates (20 x 20 cm,
1000 Microns) using appropriate mixtures of ethyl acetate and hexanes. All solvents and
reagents were purified by standard techniques reported [37] or used as supplied from
commercial sources. All compounds were named using the ACD40 Name-Pro program,
which is based on IUPAC rules. The embelin (1) used in the reactions was obtained
from Oxalis erythrorhiza Gillies ex Hook. & Arn. following the procedure described in
reference [34].
4.2 General Procedures for the preparation of dihydropyran embeline derivatives
To a mixture of 0.1 mmol of malononitrile, 0.1 mmol of the corresponding aldehyde,
and 10 mol % of Et3N in 5 mL of EtOH were added 30 mg of embelin (0.1 mmol). The
reaction mixture was stirred and refluxed until disappearance of the starting
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benzoquinone. The solvent was removed under vacuum, and the crude product was
purified by preparative-TLC to yield the corresponding dihydropyran derivative.
4.2.1 2-Amino-4-(4-chlorophenyl)-6-hydroxy-5,8-dioxo-7-undecyl-5,8-dihydro-4H-
chromene-3-carbonitrile (1).
Following the general procedure, 30 mg of embelin (0.1 mmol) was added to a mixture
of 14.3 mg of 4-chlorobenzaldehyde (0.1 mmol), 6.7 mg of malononitrile (0.1 mmol)
and 1.4 µL de Et3N (10 mol %) in 5 mL of EtOH. The reaction mixture was refluxed for
4.5 h. The solvent was removed under vacuum, and the crude product was purified by
preparative-TLC with 30% Hex/EtOAc to yield 21.1 mg (54%) of 1 as an amorphous
brown solid. Mp 154.7-155.4 °C; 1H-NMR (500 MHz CDCl3) δ 0.87 (t, J=7.0 Hz, 3H),
1.25 (bs, 16H), 1.43 (m, 2H), 2.42 (t, J=7.3 Hz, 2H), 4.58 (s, 1H), 4.94 (bs, 2H, NH2),
13
7.23 (d, J=7.9 Hz, 2H), 7.32 (d, J=7.9 Hz, 2H); C-NMR (125 MHz, CDCl3) δ 14.1
(CH3), 22.6 (CH2), 22.7 (CH2), 28.0 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6
(CH2 x 3), 31.9 (CH2), 35.6 (CH), 61.7 (C), 116.4 (C), 117.6 (C), 120.0 (C), 129.2 (CH
x 2), 129.3 (CH x 2), 134.1 (C), 139.7 (C), 147.1 (C), 151.0 (C), 157.7 (C), 179.1 (C),
181.3 (C); EIMS m/z 482 ([M+], 4), 418 (100), 294 (50), 188 (30), 154 (67); HREIMS
482.1967 (calcd. for C27H31N2O4 Cl [M+] 482.1972); IR 3360, 3185, 2925, 2853, 2290,
35
2199, 2080, 1642, 1605, 1491, 1460, 1412, 1330, 1229, 1174, 1118, 1093, 1039, 1013,
838 cm-1.
4.2.2 2-Amino-4-(4-bromophenyl)-6-hydroxy-5,8-dioxo-7-undecyl-5,8-dihydro-4H-
chromene-3-carbonitrile (2).
Following the general procedure, 30 mg of embelin (0.1 mmol) was added to a mixture
of 18.9 mg of 4-bromobenzaldehyde (0.1 mmol), 6.7 mg of malononitrile (0.1 mmol)
and 1.4 µL de Et3N (10 mol %) in 5 mL of EtOH. The reaction mixture was refluxed for
2 h. The solvent was removed under vacuum, and the crude product was purified by
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preparative-TLC with 30% Hex/EtOAc to yield 28.4 mg (54%) of 2 as an amorphous
brown solid. Mp 185.5-186.6 °C; 1H-NMR (500 MHz, CDCl3) δ 0.87 (t, J=7.3 Hz, 3H),
1.25 (bs, 16H), 1.44 (m, 2H), 2.42 (t, J=7.6 Hz, 2H), 4.56 (s, 1H), 4.93 (bs, 2H, NH2),
13
7.17 (d, J=8.5 Hz, 2H), 7.47 (d, J=8.2 Hz, 2H); C-NMR (125 MHz, CDCl3) δ 14.1
(CH3), 22.6 (CH2), 22.7 (CH2), 28.0 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6
(CH2 x 2), 29.7 (CH2), 31.9 (CH2), 35.6 (CH), 61.5 (C), 116.3 (C), 118.1 (C) 119.9 (C),
122.2 (C), 129.7 (CH x 2), 132.1 (CH x 2), 140.2 (C), 147.1 (C), 150.9 (C), 157.7 (C),
179.1 (C), 181.3 (C); EIMS m/z 427 ([M+], 10), 462 (100), 371 (75), 294 (71), 153 (55),
79
HREIMS: 526.1475 (calcd. for C27H31N2O4 Br [M+] 526.1467); IR 3423, 3365, 3187,
2922, 2851, 2388, 2294, 2198, 2114, 1986, 1912, 1642, 1605, 1536, 1488, 1462, 1411,
1330, 1228, 1118, 1075, 1006, 837 cm-1.
4.2.3 2-Amino-4-(4-fluorophenyl)-6-hydroxy-5,8-dioxo-7-undecyl-5,8-dihydro-4H-
chromene-3-carbonitrile (3).
Following the general procedure, 30 mg of embelin (0.1 mmol) was added to a mixture
of 14.3 mg of 4-fluorobenzaldehyde (0.1 mmol), 6.7 mg of malononitrile (0.1 mmol)
and 1.4 µL de Et3N (10 mol %) in 5 mL. The reaction mixture was refluxed for 20 h.
The solvent was removed under vacuum, and the crude product was purified by
preparative-TLC with 40% Hex/EtOAc to yield 31.1 mg (65%) of 3 as an amorphous
brown solid. Mp 168.4-169.2 °C. 1H-NMR (500 MHz, CDCl3) δ 0.87 (t, J=7.0 Hz, 3H),
1.25 (bs, 16H), 1.44 (m, 2H), 2.42 (t, J=7.6 Hz, 2H), 4.59 (s, 1H), 4.92 (bs, 2H, NH2),
13
7.07 (t, J=8.2 Hz, 2H), 7.26 (bs, 2H); C-NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.6
(CH2), 22.7 (CH2), 28.0 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 x 2), 29.7
(CH2), 31.9 (CH2), 35.4 (CH), 61.90 (C), 115.9 (CH x 2, J=21.5 Hz), 116.6 (C), 117.6
(C), 119.9 (C), 129.7 (CH x 2, J=8.1 Hz), 137.0 (C), 147.0 (C), 151.0 (C), 157.7 (C),
162.4 (C-F, J=240.45 Hz), 179.1 (C), 181.4 (C); EIMS m/z 466 ([M+], 35), 401 (36),
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371 (100), 232 (78), 149 (28); HREIMS 466.2268 (calcd. for C27H31N2O4F [M+]
466.2268); IR 3369, 3189, 2925, 2854, 2386, 2289, 2205, 2115, 1642, 1606, 1509,
1412, 1382, 1332, 1229, 1177, 1118, 1042, 1009, 842 cm-1.
4.2.4 2-Amino-4-(3-fluorophenyl)-6-hydroxy-5,8-dioxo-7-undecyl-5,8-dihydro-4H-
chromene-3-carbonitrile (4).
Following the general procedure, 30 mg of embelin (0.1 mmol) was added to a mixture
of 10.7 µL of 3-fluorobenzaldehide (0.1 mmol), 6.7 mg of malononitrile (0.1 mmol) and
1.4 µL de Et3N (10 mol %) in 5 mL of EtOH. The reaction mixture was refluxed for 1 h.
The solvent was removed under vacuum, and the crude product was purified by
preparative-TLC with 30% Hex/EtOAc to yield 21.5 mg (46%) of 4 as an amorphous
brown solid. Mp 167.5-168.2°C; 1H-NMR (500 MHz, CDCl3) δ 0.87 (t, J=7.0 Hz, 3H),
1.25 (bs, 16H), 1.44 (m, 2H), 2.43 (t, J=7.6 Hz, 2H), 4.60 (s, 1H), 4.93 (bs, 2H, NH2),
13
6.98 (m, 2H), 7.00 (bs, 1H), 7.10 (d, J= 7.53 Hz, 1H), 7.32 (m, 1H); C-NMR (125
MHz, CDCl3) δ 14.1 (CH3), 22.5 (CH2), 22.7 (CH2), 28.0 (CH2), 29.3 (CH2), 29.4
(CH2), 29.5 (CH2), 29.6 (CH2 x 2), 29.7 (CH2), 31.9 (CH2), 35.8 (CH), 61.6 (C), 114.9
(CH), 115.1 (CH), 115.3 (C), 116.3 (C), 117.8 (C, J= 98 Hz), 119.9 (C), 123.6 (CH,
J=3.0 Hz), 130.6 (CH, J= 7.9 Hz), 143.6 (C, J=7.6 Hz), 147.2 (C), 151.0 (C), 157.8 (C),
179.0 (C), 181.2 (C); ESMS(-) m/z 465 ([M+-1], 100), 437 (7), 399 (7), 281 (10);
HREIMS 465.2187 (calcd. for C27H30N2O4F [M+-1] 465.2190); IR 2924, 2854, 2203,
1639, 1593, 1493, 1450, 1377, 1234, 1122, 1045, 972, 894, 717, 675, 648 cm-1.
4.2.5 2-Amino-4-(3-fluoro-4-methoxyphenyl)-6-hydroxy-5,8-dioxo-7-undecyl-5,8-
dihydro-4H-chromene-3-carbonitrile (5).
Following the general procedure, 30 mg of embelin (0.1 mmol) was added to a mixture
of 15.7 mg of 3-fluor-4-methoxybenzaldehyde (0.1 mmol), 6.7 mg of malononitrile (0.1
mmol) and 1.4 µL de Et3N (10 mol %) in 5 mL of EtOH. The reaction mixture was
21

ACCEPTED MANUSCRIPT
refluxed for 20 h. The solvent was removed under vacuum, and the crude product was
purified by preparative-TLC with 30% Hex/EtOAc to yield 19 mg (38%) of 5 as an
1
amorphous brown solid. Mp 157.5-158.0 °C; H-NMR (500 MHz, CDCl3) δ 0.87 (t,
J=7.0 Hz, 3H), 1.24 (bs, 16H), 1.43 (m, 2H), 2.42 (t, J=7.9 Hz, 2H), 3.86 (s, 3H), 4.53
(s, 1H), 4.95 (bs, 2H, NH2), 6.93 (t, J=8.5 Hz, 1H), 6.99 (dd, J=11.6, 1.8 Hz, 1H), 7.04
(d, J=8.5 Hz, 1H); 13C-NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.6 (CH2), 22.7 (CH2),
28.0 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 x 2), 29.7 (CH2), 31.9
(CH2), 35.2 (CH), 56.3 (CH3), 61.6 (C), 113.5 (CH), 115.6 (CH, J=19.3 Hz), 116.4 (C),
117.7 (C), 119.9 (C), 123.9 (CH, J=3.1 Hz), 134.1 (C, J=5.0 Hz), 146.9 (C), 147.5 (C,
J=10.2 Hz), 151.0 (C), 152.4 (C-F, J=248.1 Hz), 157.7 (C), 179.2 (C), 181.4 (C); EIMS
m/z 496 ([M+], 20), 432 (37), 371 (28), 232 (43), 154 (47), 66 (100); HRMS 496.2385
(calcd. for C28H33N2O5F [M+] 496.2374); IR 3789, 3387, 3190, 2924, 2853, 2390, 2292,
2204, 1881, 1681, 1640, 1515, 1461, 1438, 1411, 1382, 1329, 1273, 1227, 1120, 1032,
977, 823 cm-1.
4.2.6 2-Amino-4-(4-cyanophenyl)-6-hydroxy-5,8-dioxo-7-undecyl-5,8-dihydro-4H-
chromene-3-carbonitrile (6).
Following the general procedure, 35 mg of embelin (0.12 mmol) was added to a mixture
of 15.6 mg of 4-formylbenzonitrile (0.12 mmol), 7.9 mg of malononitrile (0.12 mmol)
and 2 µL of Et3N (10 mol %) in 5 mL of EtOH. The reaction mixture was refluxed for 1
h. The solvent was removed under vacuum, and the crude product was purified by
preparative-TLC with 40% Hex/EtOAc to yield 50.5 mg (89%) of 6 as an amorphous
1
brown solid. Mp 176.9-178.1°C; H-NMR (500 MHz CDCl3) δ 0.87 (t, J=7.1 Hz, 3H),
1.24 (bs, 16H), 1.43 (m, 2H), 2.43 (t, J=7.8 Hz, 2H), 4.66 (s, 1H), 4.98 (bs, 2H, NH2),
13
6.92 (bs, 1H), 7.42 (d, J=8.0 Hz, 2H), 7.66 (d, J=8.0 Hz, 2H); C-NMR (125 MHz,
CDCl3) δ 14.1 (CH3), 22.6 (CH2), 22.7 (CH2), 28.0 (CH2), 29.3 (CH2), 29.5 (CH2), 29.6
22

ACCEPTED MANUSCRIPT
(CH2 x 4), 29.6 (CH2), 31.9 (CH2), 36.2 (CH), 60.8 (C), 112.1 (C), 115.7 (C), 117.3 (C),
118.3 (C), 120.3 (C), 128.8 (CH x 2), 132.9 (CH x 2), 146.2 (C), 147.4 (C), 151.0 (C),
157.9 (C), 178.8 (C), 181.1 (C); EIMS m/z 473 ([M+], 4), 409 (100), 373 (3), 268 (41),
153 (12); HREIMS 473.2342 (calcd. for C28H31N3O4 [M+] 473.2315); IR 3399, 2924,
2854, 2233, 2203, 1643, 1620, 1516, 1373, 1308, 1054, 987, 972, 852, 818 cm-1.
4.2.7 2-Amino-6-hydroxy-4-(4-nitrophenyl)-5,8-dioxo-7-undecyl-5,8-dihydro-4H-
chromene-3-carbonitrile (7).
Following the general procedure described above, 30 mg of embelin (0.1 mmol) was
added to a mixture of 15.4 mg of 4-nitrobenzaldehyde (0.1 mmol), 6.7 mg of
malononitrile (0.1 mmol) and 1.4 µL de Et3N (10% mol) in 5 mL of EtOH. The reaction
mixture was refluxed for 1.5 h. The solvent was removed under vacuum, and the crude
product was purified by preparative-TLC with 30% Hex/EtOAc to yield 47 mg (94%)
1
of 7 as an amorphous brown solid. Mp 147.9-148.3 °C; H-NMR (500 MHz, CDCl3) δ
0.87 (t, J= 6.8 Hz, 3H), 1.24 (bs, 16H), 1.43 (m, 2H), 2.43 (t, J= 7.5 Hz, 2H), 4.71 (s,
1H), 5.07 (bs, 2H, NH2), 6.94 (bs, 1H, OH), 7.48 (d, J=8.5 Hz, 2H), 8.21 (d, J=8.5 Hz,
13
2H); C-NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.6 (CH2), 22.7 (CH2), 28.0 (CH2),
29.3 (CH2 x 2), 29.5 (CH2 x 2), 29.6 (CH2 x 2), 31.9 (CH2), 36.0 (CH), 60.6 (C), 115.6
(C), 117.4 (C), 120.3 (C), 124.3 (CH x 2), 129.0 (CH x 2 ), 147.5 (C), 147.6 (C), 148.0
(C), 151.0 (C), 158.0 (C), 178.8 (C), 181.2 (C); EIMS m/z (%) 430 ([M+-NO2,-OH],
31), 429 (100), 289 (44), 154 (30); ESHRMS(-) 492.2143 (calcd. for C27H30N3O6 [M-
H]+ 492.2135; IR 3393, 3196, 2927, 2856, 2293, 2206, 2119, 1934, 1646, 1609, 1522,
1348, 1235, 1117, 1048, 979, 863, 834 cm-1.
4.2.8 2-Amino-6-hydroxy-4-(3-nitrophenyl)-5,8-dioxo-7-undecyl-5,8-dihydro-4H-
chromene-3-carbonitrile (8).
23

ACCEPTED MANUSCRIPT
Following the general procedure, 33 mg of embelin (0.11 mmol) was added to a mixture
of 16.8 mg of 3-nitrobenzaldehyde (0.11 mmol), 7.4 mg of malononitrile (0.11 mmol)
and 2 µL de Et3N (10 mol %) in 5 mL of EtOH. The reaction mixture was refluxed for 1
h. The solvent was removed under vacuum, and the crude product was purified by
preparative-TLC with 40% Hex/EtOAc to yield 50.5 mg (91%) of 8 as an amorphous
1
brown solid. Mp 213.1-214.8°C; H-NMR (500 MHz CDCl3) δ 0.88 (t, J=7.1 Hz, 3H),
1.25 (bs, 16H), 1.44 (m, 2H), 2.44 (t, J=7.7 Hz, 2H), 4.74 (s, 1H), 5.03 (bs, 2H, NH2),
6.92 (bs, 1H, OH), 7.55 (t, J=7.8 Hz, 1H), 7.69 (d, J=7.6 Hz, 1H), 8.13 (t, J= 1.8 Hz,
13
1H), 8.16 (dd, J= 1.9, 8.2Hz, 1H); C-NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.6
(CH2), 22.7 (CH2), 28.0 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 x 2), 29.7
(CH2), 31.9 (CH2), 36.0 (CH), 60.4 (C), 115.6 (C), 117.4 (C), 120.3 (C), 123.0 (CH),
123.2 (CH), 129.9 (CH), 134.4 (CH), 143.3 (C), 147.4 (C), 148.7 (C), 151.0 (C), 158.0
(C), 178.8 (C), 181.2 (C); EIMS m/z 493 ([M+], 28), 429 (100), 371 (43), 294 (37), 153
(41); HREIMS 493.2198 (calcd. for C27H31N3O6 [M+] 493.2213); IR 3387, 3337, 2924,
2854, 1643, 1609, 1524, 1504, 1381, 1346, 1288, 1057, 987, 972, 822, 667 cm-1.
4.2.9 2-Amino-6-hydroxy-5,8-dioxo-4-(4-(trifluoromethyl)phenyl)-7-undecyl-5,8-
dihydro-4H-chromene-3-carbonitrile (9).
Following the general procedure, 33 mg of embelin (0.1 mmol) was added to a mixture
of 15.3 µL of 4-(trifluoromethyl)benzaldehyde (0.1 mmol), 7.4 mg of malononitrile (0.1
mmol) and 2 µL de Et3N (10 mol %) in 5 mL of EtOH. The reaction mixture was
refluxed for 1.5 h. The solvent was removed under vacuum, and the crude product was
purified by preparative-TLC with 40% Hex/EtOAc to yield 47.1 mg (81%) of 9 as an
1
amorphous brown solid. Mp 164.8-166.0°C; H-NMR (500 MHz CDCl3) δ 0.88 (t,
J=7.4 Hz, 3H), 1.25 (bs, 16H), 1.44 (m, 2H), 2.43 (t, J=7.7 Hz, 2H), 4.67 (s, 1H), 4.98
13
(bs, 2H, NH2), 6.95 (bs, 1H, OH), 7.42 (d, J=7.8 Hz, 2H), 7.62 (d, J=8.2 Hz, 2H); C-
24

ACCEPTED MANUSCRIPT
NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.6 (CH2), 22.7 (CH2), 28.0 (CH2), 29.3 (CH2),
29.4 (CH2), 29.5 (CH2), 29.6 (CH2 x 2), 29.7 (CH2), 31.9 (CH2), 35.9 (CH), 61.2 (C),
116.1 (C), 117.5 (C), 120.1 (C), 123.8 (C, J= 275.4 Hz), 126.0 (CH x 2, J=3.4 Hz),
128.4 (CH x 2), 130.3 (C, J= 32.7Hz), 145.0 (C), 147.3 (C), 151.0 (C), 157.8 (C), 178.9
(C), 181.2 (C); ESHRMS(-) 515.2157 (calcd. for C28H30N2O4F3 [M+-1] 515.2158); IR
3398, 2924, 2854, 1670, 1620 1500, 1408, 1377, 1323, 1064, 972, 875, 806, 737, 667
cm-1.
4.2.10 4-(2-Amino-3-cyano-6-hydroxy-5,8-dioxo-7-undecyl-5,8-dihydro-4H-
chromen-4-yl)benzoic acid (10).
Following the general procedure, 30 mg of embelin (0.1 mmol) was added to a mixture
of 15.3 mg of 4-formylbenzoic acid (0.1 mmol), 6.7 mg of malononitrile (0.1 mmol) in
5 mL of EtOH. The reaction mixture was refluxed for 1 h. The solvent was removed
under vacuum, and the crude product was purified by preparative-TLC with 40%
Hex/EtOAc to yield 40.1 mg (80%) of 10 as an amorphous brown solid. Mp 198.1-
199.8°C; H-NMR (500 MHz, DMSO-d6) δ 0.86 (t, J=6.7 Hz, 3H), 1.23 (bs, 16H), 1.32
(m, 2H), 2.26 (t, J=7.7 Hz, 2H), 4.51 (s, 1H), 7.28 (bs, 2H, NH2), 7.38 (d, J=7.9 Hz,
2H), 7.89 (d, J= 7.9 Hz, 2H); 13C-NMR (150 MHz, DMSO-d6) δ 13.9 (CH3), 22.1 (CH2
x 2), 28.4 (CH2), 28.7 (CH2), 29.0 (CH2), 29.1 (CH2 x 2), 29.2 (CH2), 29.3 (CH2), 31.3
(CH2), 35.9 (CH), 57.0 (C), 78.9 (C), 114.6 (C), 119.5 (C), 127.6 (CH x 2), 128.1 (C),
129.2 (C), 129.6 (CH x 2), 146.6 (C), 149.0 (C), 158.9 (C), 167.1 (C); ESMS(-) m/z 491
([M+-1], 100), 425 (50), 413 (28), 281 (35), 245 (60); ESHRMS 491.2181 (calcd. for
C28H31N2O6 (M+-1) 491.2182); IR 3518, 3398, 1682, 1666, 1620 1493, 1412, 1381,
1053, 978, 798, 667 cm-1.
4.2.11 Methyl 4-(2-amino-3-cyano-6-hydroxy-5,8-dioxo-7-undecyl-5,8-dihydro-4H-
chromen-4-yl)benzoate (11).
25

ACCEPTED MANUSCRIPT
Following the general procedure, 30 mg of embelin (0.1 mmol) was added to a mixture
of 14.8 mg of methyl 4-formylbenzoate (0.1 mmol), 6.7 mg of malononitrile (0.1 mmol)
and 1.4 µL de Et3N (10 mol %) in 5 mL of EtOH. The reaction mixture was refluxed for
2 h. The solvent was removed under vacuum, and the crude product was purified by
preparative-TLC with 40% Hex/EtOAc to yield 38.6 mg (75%), of 11 as an amorphous
1
brown solid. Mp 146.6-147.9°C; H-NMR (500 MHz CDCl3) δ 0.87 (t, J=7.0 Hz, 3H),
1.24 (bs, 16H), 1.43 (m, 2H), 2.42 (t, J=7.9 Hz, 2H), 3.90 (s, 3H), 4.65 (s, 1H), 4.69 (bs,
2H, NH2), 6.94 (bs, 1H, OH), 7.37 (d, J=8.1 Hz, 2H), 8.01 (d, J=8.3 Hz, 2H); 13C-NMR
(125 MHz, CDCl3) δ 14.1 (CH3), 22.6 (CH2), 22.7 (CH2), 28.0 (CH2), 29.3 (CH2), 29.4
(CH2), 29.5 (CH2), 29.6 (CH2 x 2), 29.7 (CH2), 31.9 (CH2), 36.0 (CH), 52.1 (CH3), 61.3
(C), 116.2 (C), 117.5 (C), 119.1 (C), 128.0 (CH x 2), 129.9 (C), 130.3 (CH x 2), 146.0
(C), 147.3 (C), 151.0 (C), 157.8 (C), 166.5 (C), 179.0 (C), 181.2 (C); ESHRMS(-)
505.2336 (calcd. for C29H33N2O6 [M+-1] 505.2339); IR 3399, 2924, 2854, 2198, 1682,
1616, 1447, 1380, 1284, 1064, 968, 667 cm-1
.
4.2.12
2-Amino-6-hydroxy-5,8-dioxo-4-phenyl-7-undecyl-5,8-dihydro-4H-
chromene-3-carbonitrile (12).
Following the general procedure described above, 30 mg of embelin (0.1 mmol) was
added to a mixture of 10.8 mg of benzaldehyde (10.4 µL, 0.1 mmol), 6.7 mg of
malononitrile (0.1 mmol) and 1.4 µL de Et3N (10 mol %) in 5 mL of EtOH. The
reaction mixture was refluxed for 1.5 h. The solvent was removed under vacuum, and
the crude product was purified by preparative-TLC with 30% Hex/EtOAc to yield 13.6
1
mg (30%) of 12 as an amorphous brown solid. Mp 155.9-156.6 °C; H-NMR (500
MHz, CDCl3) δ 0.87 (t, J=7.3 Hz, 3H), 1.24 (bs, 16H), 1.43 (m, 2H), 2.42 (t, J=7.6 Hz,
2H), 4.59 (s, 1H), 4.91 (bs, 2H, NH2), 7.27 (m, 3H), 7.34 (m, 2H); 13C-NMR (125 MHz,
CDCl3) δ 14.1 (CH3), 22.6 (CH2), 22.7 (CH2), 28.0 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5
26

ACCEPTED MANUSCRIPT
(CH2), 29.6 (CH2 x3), 31.9 (CH2), 36.0 (CH), 62.1 (C), 116.8 (C), 119.8 (C), 127.9 (CH
x 2), 128.1 (CH), 129.0 (CH x 2), 141.2 (C), 147.1 (C), 151.0 (C), 157.7 (C), 179.3 (C),
181.4 (C); EIMS m/z 448 ([M+], 20), 384 (39), 371 (100), 214 (32); HREIMS 448.2357
(calcd. for C27H32N2O4 [M+] 448.2362); IR 3373, 2918, 2851, 2203, 1680, 1641, 1607,
1496, 1454, 1382, 1330, 1226, 1175, 1117, 998, 835 cm-1.
4.2.13 2-Amino-6-hydroxy-4-(4-methoxyphenyl)-5,8-dioxo-7-undecyl-5,8-dihydro-
4H-chromene-3-carbonitrile (13).
Following the general procedure, 30 mg of embelin (0.1 mmol) was added to a mixture
of 13.9 mg of 4-methoxybenzaldehyde (12.5 µL, 0.1 mmol), 6.7 mg of malononitrile
(0.1 mmol) and 1.4 µL de Et3N (10 mol %) in 5 mL of EtOH. The reaction mixture was
refluxed for 9 h. The solvent was removed under vacuum, and the crude product was
purified by preparative-TLC with 30% Hex/EtOAc to yield 22.9 mg (47%) of 13 as an
1
amorphous brown solid. Mp 154.1-154.6 °C; H-NMR (500 MHz, CDCl3) δ 0.88 (t,
J=7.0 Hz, 3H), 1.25 (bs, 16H), 1.43 (m, 2H), 2.42 (t, J=7.9 Hz, 2H), 3.78 (s, 3H), 4.54
13
(s, 1H), 4.85 (bs, 2H, NH2), 6.86 (d, J=8.5 Hz, 2H), 7.20 (d, J=8.5 Hz, 2H); C-NMR
(125 MHz, CDCl3) δ 14.1 (CH3), 22.5 (CH2), 22.7 (CH2), 28.0 (CH2), 29.3 (CH2), 29.4
(CH2), 29.5 (CH2), 29.6 (CH2 x 2), 29.7 (CH2), 31.9 (CH2), 35.2 (CH), 55.3 (CH3), 66.4
(C), 114.4 (CH x 2), 116.9 (C), 117.9 (C), 119.7 (C), 129.1 (CH x 2), 133.4 (C), 146.8
(C), 150.9 (C), 157.5 (C), 159.4 (C), 179.3 (C), 181.5 (C); EIMS m/z (%) 478 ([M+],
0.3), 414 (100), 274 (20), 147 (19); HREIMS 478.2475 (calcd. for C28H34N2O5 [M+]
478.2468); IR 3356, 3188, 2923, 2853, 2388, 2290, 2202, 2079, 1897, 1681, 1641,
1608, 1511, 1462, 1411, 1382, 1331, 1230, 1176, 1118, 1037, 836 cm-1.
4.2.14 2-amino-4-(3,4-dimethoxyphenyl)-6-hydroxy-5,8-dioxo-7-undecyl-5,8-
dihydro-4H-chromene-3-carbonitrile (14).
27

ACCEPTED MANUSCRIPT
Following the general procedure, 30 mg of embelin (0.1 mmol) was added to a mixture
of 17 mg of 3,4-dimethoxybenzaldehyde (0.1 mmol), 6.7 mg of malononitrile (0.1
mmol) and 1.4 µL de Et3N (10 mol %) in 5 mL of EtOH. The reaction mixture was
refluxed for 4 h. The solvent was removed under vacuum, and the crude product was
purified by preparative-TLC with 40% Hex/EtOAc to yield 33.5 mg (65%) of 14 as an
1
amorphous brown solid. Mp 150.2-151.9°C; H-NMR (500 MHz, CDCl3) δ 0.87 (t,
J=7.0 Hz 3H), 1.25 (bs, 16H), 1.44 (m, 2H), 2.43 (t, J=7.8 Hz, 2H), 3.85 (s, 3H), 3.89
(s, 3H), 4.54 (s, 1H), 4.90 (bs, 2H, NH2); 6.81 (m, 3H); 13C-NMR (125 MHz, CDCl3) δ
14.1 (CH3), 22.6 (CH2), 22.7 (CH2), 28.0 (CH2), 29.3 (CH2), 29.4 (CH2), 29.6 (CH2 x 2),
29.7 (CH2), 31.9 (CH2), 35.6 (CH), 55.9 (CH3), 56.0 (CH3), 62.2 (C), 111.3 (CH), 111.4
(CH), 116.8 (C), 117.9 (C), 119.1 (C), 119.7 (C), 120.1 (CH), 133.8 (C), 146.8 (C),
148.9 (C), 149.2 (C), 151.0 (C), 157.6 (C), 179.4 (C), 181.5 (C); EIMS m/z 508 ([M+],
43), 444 (31), 414 (33), 294 (68), 153 (100); HREIMS 508.2558 (calcd. for C29H36N2O6
[M+] 508.2573); IR 3360, 3797, 3005, 2924, 2877, 2368, 2198, 2168, 1978, 1732,
1643, 1516, 1462, 1327, 1254, 1231, 1126, 1026, 883, 818, 675 cm-1.
4.2.15
2-Amino-4-(benzo[d][1,3]dioxol-5-yl)-6-hydroxy-5,8-dioxo-7-undecyl-5,8-
dihydro-4H-chromene-3-carbonitrile (15).
Following the general procedure, 30 mg of embelin (0.1 mmol) was added to a mixture
of 15.3 mg of piperonal (0.1 mmol), 6.7 mg of malononitrile (0.1 mmol) and 1.4 µL de
Et3N (10 mol %) in 5 mL of EtOH. The reaction mixture was refluxed for 9 h. The
solvent was removed under vacuum, and the crude product was purified by preparative-
TLC with 5% DCM/EtOAc to yield 10.7 mg (22%) of 15 as an amorphous brown solid.
1
Mp 126.5-127.2 °C; H-NMR (500 MHz, CDCl3) δ 0.87 (t, J=7.0 Hz 3H), 1.25 (bs,
16H), 1.44 (m, 2H), 2.42 (t, J=7.6 Hz, 2H), 4.51 (s, 1H), 4.91 (bs, 2H, NH2), 5.95 (d,
13
J=0.9 Hz, 2H), 6.75 (m, 3H); C-NMR (125 MHz, CDCl3) δ 14.1 (CH3), 22.6 (CH2),
28

ACCEPTED MANUSCRIPT
22.7 (CH2), 28.0 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2 x 2), 29.7 (CH2),
31.9 (CH2), 35.7 (CH), 62.2 (C), 101.3 (CH2), 108.3 (CH), 108.5 (C), 108.6 (CH), 116.7
(C), 119.8 (C), 121.5 (CH), 135.2 (C), 146.9 (C), 147.4 (C), 148.2 (C), 151.0 (C), 157.6
(C), 179.2 (C), 181.4 (C); EIMS m/z 492.5 ([M+], 3), 216 (100), 172 (11), 114 (23), 69
(14); HRMS: 492.2274 (calcd. for C28H32N2O6 [M+] 492.2260); IR 3363, 3188, 2923,
2854, 2205, 1681, 1646, 1605, 1503, 1447, 1412, 1389, 1333, 1232, 1118, 1041, 1001,
928, 864 cm-1.
4.2.16 2-Amino-6-hydroxy-5,8-dioxo-4-(pyridin-3-yl)-7-undecyl-5,8-dihydro-4H-
chromene-3-carbonitrile (16).
Following the general procedure, 33 mg of embelin (0.1 mmol) was added to a mixture
of 10.5 µL of 3-pyridinecarboxaldehyde (0.1 mmol) and 7.4 mg of malononitrile (0.1
mmol) in 5 mL of ethanol and 2 µL de Et3N (10% mol). The reaction mixture was
refluxed for 1 h. The solvent was removed under vacuum, and the crude product was
purified by preparative-TLC with 40% Hex/EtOAc to yield 37.1 mg (74%) of 16 as an
1
amorphous brown solid. Mp 159.7-161.0°C; H-NMR (500 MHz CDCl3) δ 0.86 (t,
J=7.1 Hz, 3H), 1.24 (bs, 16H), 1.44 (m, 2H), 2.44 (t, J=7.6 Hz, 2H), 4.79 (s, 1H), 5.19
(bs, 2H, NH2), 7.35 (dd, J= 4.9, 7.9 Hz, 1H), 7.67 (dt, J=1.9, 7.8 Hz, 1H), 8.44 (d, J=4.3
13
Hz, 1H), 9.02 (s, 1H); C-NMR (150 MHz, CDCl3) δ 14.1 (CH3), 22.6 (CH2), 22.7
(CH2), 28.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2 x 2),
31.9 (CH2), 33.8 (CH), 59.6 (C), 116.0 (C), 117.8 (C), 120.4 (C), 124.3 (CH), 136.6
(CH), 137.5 (C), 146.6 (CH), 147.8 (C), 149.7 (CH), 153.9 (C), 158.9 (C), 179.2 (C),
181.6 (C); ESMS m/z 449 ([M+], 30), 448 (100), 329 (8), 255 (10), 212 (22); HRMS
448.2227 (calcd. for C26H30N3O4 [M+] 448.2236); IR 3425, 3402, 3174, 2924, 2854,
2203, 1674, 1635, 1597, 1500, 1327, 1231, 1115, 1045, 806, 756, 709 cm-1.
29