Structure-activity relationship (SAR) study of ethyl 2-amino-6-(3,5- dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017) and the potential of the lead against multidrug resistance in cancer treatment

Article abstract of DOI:10.1021/jm300515q

Multidrug resistance (MDR) against standard therapies poses a serious challenge in cancer treatment, and there is a clinical need for new anticancer agents that would selectively target MDR malignancies. Our previous studies have identified a 4H-chromene system, CXL017 (4) as an example, that can preferentially kill MDR cancer cells. To further improve its potency, we have performed detailed structure-activity relationship (SAR) studies at the 3, 4, and 6 positions of the 4H-chromene system. The results reveal that the 3 and 4 positions prefer rigid and hydrophobic functional groups while the 6 position prefers a meta or para-substituted aryl functional group and the substituent should be small and hydrophilic. We have also identified and characterized nine MDR cancer cells that acquire MDR through different mechanisms and demonstrated the scope of our new lead, 9g, to selectively target different MDR cancers, which holds promise to help manage MDR in cancer treatment.

Full text of DOI:10.1021/jm300515q

Article
pubs.acs.org/jmc
Structure−Activity Relationship (SAR) Study of Ethyl 2-Amino-6-(3,5-
dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-
carboxylate (CXL017) and the Potential of the Lead against
Multidrug Resistance in Cancer Treatment
Gopalakrishnan Aridoss, Bo Zhou, David L. Hermanson, Nicholas P. Bleeker, and Chengguo Xing*
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, 308 Harvard Street SE, Minneapolis, Minnesota
55455, United States
S
* Supporting Information
ABSTRACT: Multidrug resistance (MDR) against standard therapies poses a serious challenge in cancer treatment, and there is
a clinical need for new anticancer agents that would selectively target MDR malignancies. Our previous studies have identified a
4H-chromene system, CXL017 (4) as an example, that can preferentially kill MDR cancer cells. To further improve its potency,
we have performed detailed structure−activity relationship (SAR) studies at the 3, 4, and 6 positions of the 4H-chromene system.
The results reveal that the 3 and 4 positions prefer rigid and hydrophobic functional groups while the 6 position prefers a meta or
para-substituted aryl functional group and the substituent should be small and hydrophilic. We have also identified and
characterized nine MDR cancer cells that acquire MDR through different mechanisms and demonstrated the scope of our new
lead, 9g, to selectively target different MDR cancers, which holds promise to help manage MDR in cancer treatment.
localized calcium channel/pump, and p-glycoprotein in MDR
with intracellular Ca2+ as a possible link.
INTRODUCTION
■
Multidrug resistance (MDR) is a general phenomenon among
all malignancies. Anticancer agents that can circumvent MDR
are highly needed to achieve effective treatment. One major
mechanism in acquiring MDR is overexpression of the
antiapoptotic Bcl-2 family proteins, which occurs in 60−90%
of all types of cancers.1 Such overexpression is proposed as one
general MDR mechanism because many standard cancer
therapies introduce their activity via activation of the apoptotic
machinery.2,3 The other major mechanism is overexpression of
ATP-binding cassette (ABC) transporter proteins, such as p-
glycoprotein.4,5 The overexpressed ABC transporter proteins
decrease the concentration of anticancer agents in tumor cells
through enhanced drug efflux, leading to MDR.
The Bcl-2 family proteins regulate apoptosis through both
the mitochondrial and the endoplasmic reticulum (ER)
pathways.6,7 The ER pathway communicates with the
mitochondrial pathway via control of intracellular Ca2+
distribution.8 Mechanistically, the antiapoptotic Bcl-2 family
proteins on the ER physically interact with inositol triphosphate
receptor (IP3R)9−11 or sarco/endoplasmic reticulum Ca2+-
ATPase (SERCA),12,13 two key ER-localized Ca2+ channel/
pump, to regulate Ca2+ homeostasis. Studies also suggest that
modulating intracellular Ca2+ may affect the activity of p-
glycoprotein.14−16 These lines of evidence suggest that there
may be communications among the Bcl-2 family proteins, ER-
Significant progress has been achieved over the past decades
toward the development of anticancer agents that may
overcome MDR via inhibition of the antiapoptotic Bcl-2 family
proteins or the ABC transporter proteins. For instance, ABT-
737 (1) and its analogue ABT-263 (2) have been developed as
inhibitors against Bcl-2 and Bcl-XL proteins (Figure 1), aiming
to reactivate the apoptotic machinery in cancer cells, and
candidate 2 has revealed some anticancer effects in recent
clinical trials.17,18 These drug candidates have also shown
potential to treat MDR cancers.19,20 Similarly, various small-
molecule modulators for ABC transporter proteins have been
developed and some of them have demonstrated promise to
nullify MDR in different cancer models. In particular, the recent
work from Gottesman’s group explored and demonstrated the
feasibility of selectively targeting p-glycoprotein based MDR
cancer cells.21,22 One lead candidate, isatin-β-thiosemicarba-
zone (3, Figure 1), reveals increased cytotoxicity, namely,
collateral sensitivity, toward p-glycoprotein based MDR cancer
cells. Nonetheless, no anticancer candidates have been designed
to simultaneously target both MDR mechanisms, which may be
Received: April 11, 2012
Published: May 14, 2012
© 2012 American Chemical Society
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Figure 1. Structures of the representative inhibitors for antiapoptotic Bcl-2 family proteins (1, 2, and 5), p-glycoprotein (3), SERCA (6), and one
CXL candidate (4).
a
Scheme 1. Synthesis of Series 7
a
Reagents and conditions: (i) 3,5-dimethoxyphenylboronic acid, Pd(OAc)2, TPP, DME/H2O (1:1), K2CO3, rt, 24 h; (ii) DMA, POCl3, DCM, 80
°C, 4 h; NaHCO3, 60 °C, 1 h; (iii) 3,5-dimethoxyphenylboronic acid, Pd(PPh3)4, toluene/H2O (8:2), K2CO3, 80 °C, overnight; (iv) methyl
cyanoacetate or ethyl cyanoacetate, R1OH, NaOR1, rt, 2 h; (v) NaBH4, MeOH, rt, 5 h; (vi) ethyl cyanoacetate, allyl alcohol, Na, rt, 2 h; (vii) m-
CPBA, DCM, 0 °C to rt, 24 h; (viii) malononitrile, EtOH, NaOEt, rt, 3 h; (ix) benzene, NEt3, reflux, 6 h.
necessary, since cancer cells can acquire their MDR through
multiple mechanisms simultaneously.
comparison to 5, 4 and its analogues have significantly
improved stability with some of them having improved
potency.27−30 Genetically overexpressing antiapoptotic Bcl-2
or Bcl-XL protein fails to introduce resistance to CXL
candidates, although such overexpression confers significant
MDR to standard cancer therapies.28 More importantly, five
natural MDR cancer cell lines show no resistance to CXL
candidates.29,30 Some of them actually demonstrate collateral
sensitivity to CXL candidates. Results of our mechanistic
We have recently developed a series of CXL candidates,
CXL017 (4) as one example, based on HA 14-1 (5), a putative
Bcl-2 inhibitor (Figure 1).23 Candidate 5 was selected as our
initial lead because of its ability to selectively kill cancer cells
with elevated antiapoptotic Bcl-2 protein.24 Candidate 5 also
modulates the intracellular Ca2+ homeostasis25 and synergizes
with various cancer therapies in different cancer models.26 In
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a
Scheme 2. Synthesis of Series 8
a
Reagents and conditions: (i) R-B(OH)2 or R-B(OH)2 MIDA ester, Pd(OAc)2 or Pd(PPh3)4, suitable base and solvent, 80 °C, overnight; (ii) DMA,
POCl3, DCM, 80 °C, 4 h; NaHCO3, 60 °C, 1 h; (iii) ethyl cyanoacetate, EtOH, NaOEt, rt, 2 h.
investigation suggest that CXL candidates cause ER Ca2+
release and induce apoptosis mainly through the ER pathway
via antagonizing the antiapoptotic Bcl-2 family proteins and
SERCA protein.31 Given the potential communications
between antiapoptotic Bcl-2 family proteins and p-glycoprotein
via ER calcium pump/channel and Ca2+ homeostasis, we
hypothesize that CXL candidates can effectively target MDR
introduced by both mechanisms in cancer treatment. Indeed,
our preliminary data show that candidate 4 effectively inhibits
the transporting activity of p-glycoprotein in whole cells, and
the mechanism of such inhibition is currently under
investigation (unpublished results).
Nonetheless, our lead candidate demonstrates relatively weak
potency against parental cancer cell lines, limiting their in vivo
evaluation. In order to improve the potency of our lead
candidates, we have performed additional structural modifica-
tions at the 3, 4, and 6 positions of the 4H-chromene system.
This report summarizes our sharp SAR results at these
positions and the identification of several candidates with
submicromolar potency. We have also characterized nine MDR
cancer cells and evaluated the potential of 9g, our new lead
candidate, to overcome MDR in comparison to several drug
candidates that target antiapoptotic Bcl-2 family proteins (1), p-
glycoprotein (3), or SERCA (thapsigargin, 6).
at the 3 position (7l) from the corresponding nitrile (7k) was
achieved via 1,3-dipolar cycloaddition reaction with nitrile oxide
generated in situ from 4-methoxyphenylhydroximinoyl chlor-
ide33 in the presence of triethylamine.34
We next introduced fluorine substituted aryls or heterocyclic
aromatic systems at the 6 position (series 8). As outlined in
Scheme 2, each candidate was obtained starting from 5-
bromosalicylaldehyde through three steps: Suzuki coupling,
cyclization to coumarin, and chromene formation. The
sequence of these three steps and the conditions of Suzuki
coupling reaction vary for different analogues because a
universal protocol that would work for all of these analogues
could not be found because of different reactivity among the
substrates and ease of purification. The Suzuki reactions with
heterocyclic counterparts were achieved through Burke’s
modification.35
Results from our previous studies also indicate that the meta
and para positions of the 6-phenyl group are critical to the
potency of CXL candidates: functionalizing each of these
positions with a methoxy or a methyl improved their
cytotoxicity, while functionalizing both positions led to
significant loss of activity. In this study, we prepared a panel
of primary, secondary, and tertiary amino candidates at these
positions to further define the functional group preference,
including size and hydrophilicity (series 9, Scheme 3). Suzuki
coupling of a nitro-substituted arylboronic acid with 5-
bromocoumarin followed by Pd/C-catalyzed reduction fur-
nished the desired aminocoumarins, which in turn were
converted to the target 4H-chromenes with ethanol or
propargyl alcohol. It is worth mentioning that the nature of
the alcohols used for the synthesis of 4H-chromene from 6-
arylcoumarin greatly influences the conversion rate, the final
product(s), and their yields. Unlike ethanol, where the yields
are relatively good and the reaction rate is relatively fast,
reaction with propargyl alcohol is very moisture sensitive and
slow with low yield. As expected, in situ base catalyzed
transesterification occurred when propargyl alcohol was used.
Indeed, transesterification is relatively fast that dipropargyl
derivative is the major product with minimal or no
monopropargyl counterpart. With the support of 2D NMR
(see Supporting Information Figures S1 and S2), it was
confirmed that the C-4 ester bears the propargyl group in the
monopropargyl analogue, consistent with its reaction mecha-
nism.36 For the preparation of secondary and tertiary amino
RESULTS AND DISCUSSION
■
Chemistry. We prepared the 4H-chromene analogues
according to the procedures adopted from our previous work
with slight modifications (Schemes 1−3).29,30
Results from our previous work suggest that the potency of
CXL candidates is very sensitive to the functional groups at the
3 and 4 positions. In this study we further characterized the
preferred property of the functional groups at these positions,
including size, lipophilicity, and flexibility (Scheme 1, series 7).
Of the two routes to 6-(3′,5′-dimethoxyphenyl)coumarin,
Suzuki−Miyaura coupling was carried out with 6-bromocou-
marin32 instead of 5-bromosalicylaldehyde, as the former route
gave higher percentage of conversion along with significantly
lower dimer formation derived from the boronic acid and easier
purification. To explore the impact of lipophilicity, the diallylic
analogue (7f) was converted to the dioxirane methyl derivative
(7j) via standard epoxidation. 7j can be compared with the
dicyclopropylmethyl candidate (7i) given their similarity in size,
shape, and flexibility. Introduction of an oxadiazole ring system
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a
Scheme 3. Synthesis of Series 9
a
Reagents and conditions: (i) ArB(OH)2, Pd(PPh3)4, K2CO3, toluene/H2O (8:2), 90 °C, overnight (for 11k, (3-carbamoylphenyl)boronic acid
MIDA ester and dioxane−water were used); (ii) ECA, R1OH, NaOR1, rt, 2−12 h; (iii) Pd/C (10%), HCOONH4, MeOH, rt, 6 h; (iv) malononitrile,
dry propargyl alcohol, Na, rt, 12 h; (v) ICH2CH2OH, ACN, 80 °C, overnight; (vi) ClCH2COCl, NEt3, dry DCM, rt, 2 h; (vii) ethylammonium
nitrate ionic liquid, CH(OEt)3, TMSN3, rt, 1 h; (viii) RI, NaH, Dry DMF, rt, 8 h; (ix) MeI (5 equiv), NaH (2.5 equiv), dry DMF, rt, 2 h; (x) TFA,
dry DCM, rt, 6 h; (xi) MeI, NaH, dry DMF, rt, 2 h.
analogues, direct alkylation of the primary amine was not
successful in some cases. Under such circumstances, we started
with the N-Boc-protected arylboronic acid to furnish the N-Boc
protected 4H-chromene, which through conventional protocol
led to the desired N-alkylated derivatives in moderate to good
yield. Interestingly, attempted methylation of N-Boc protected
amine (11n) with excess molar ratio of MeI and NaH led to the
cleavage of the 4H-chromene to furnish 11r1,37 whereas the
equivalent molar ratio yielded the N-methylated derivative as
the major product with minor amount of 11r1. The ring
cleavage reaction was not noticed when ethyl iodide was used
that it gave solely the desired product. N,N-Dimethylated
analogues were obtained by further methylation of the
corresponding N-methylated analogues. The amino analogue
(9c) was also functionalized to provide compounds 9n and 9o
by following earlier reports,38 whereas 1H-1,2,3,4-tetrazole (9q)
was conveniently synthesized in one-pot from 9c by reaction
with TMSN3 and CH(OEt)3 using the readily available,
recyclable, Brønsted acidic ionic liquids [EtNH3][NO3].39
In Vitro Cytotoxicity in HL60. The cytotoxic potency of
these candidates was first evaluated in HL60 following
established procedures,29 given that HL60 parental line is
generally less sensitive to CXL candidates compared to HL60/
MX2, an MDR resistant cell line developed from HL60.30 The
cytotoxicity results of series 7 (Table 1) demonstrate that the
functional groups at the 3 and 4 positions are important. There
is a 13-fold difference in cytotoxicity between the methyl (7a)
and the n-butyl (7e) analogues with the ethyl (7c) and n-propyl
(7d) analogues in between. Reduction in flexibility at these
positions improves activity. This is reflected by the dipropargyl
esters (7g) being 3 and 5 times more potent than the diallyl
(7f) and di-n-propyl (7d) candidates, respectively. Similarly, the
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Table 1. IC50 (μM) of Analogues 7a−l in HL60
a
compd
R1
R2
IC50 SEM
7a
Me
Me
2.42 0.77
10.8 0.5
10.7 0.5
7.6 0.3
7b
Et
Me
7c29
7d29
7e29
7f29
7g29
7h29
7i29
7j
Et
Et
n-Pr
n-Bu
allyl
C3H3
C3H3
n-Pr
n-Bu
allyl
C3H3
Et
32
2
4.7 0.3
1.5 0.1
4.7 0.2
6.7 0.5
>80
cyclopropylmethyl
oxirane methyl
cyclopropylmethyl
oxirane methyl
7k
19.6 3.4
>80
7l
a
Results are given as the mean of at least two independent experiments with triplicates in each experiment.
cyclopropylmethyl analogue (7i) is more potent than the n-
butyl analogue (7e). Incorporation of a cyano functional group
at the 3 position (7k), which is the least flexible and the
smallest in size, however, leads to significant loss of activity
relative to 7c. This suggests that the substitution at the 3
position cannot be too small or that the lipophilicity of the
functional group is important as well. Converting the cyano
group into an oxadiazole (7l) results in complete loss of
activity, likely due to increased hydrophilicity or size. In
comparison to the cyclopropylmethyl candidate, the oxirane
methyl substitution (7j) is completely inactive, confirming that
the 3 and 4 positions favor lipophilic functional groups over
hydrophilic functional groups. Overall, the alkoxy groups on the
3 and 4 esters prefer to be lipophilic and rigid with a size of one
to three carbons.
heterocyclic analogues (Table 3), 4′-pyridinyl shows the
greatest improvement over sHA 14-1 and the position of the
nitrogen in the pyridine ring is important; the 4′-pyridinyl
candidate (8k) is 2- and 8-fold more potent than the 3′-
pyridinyl (8j) and 2′-pyridinyl (8i) candidates, respectively. As
our previous SAR showed that introduction of a methoxy group
at the 3′ position significantly improved the potency, we have
designed and evaluated analogue 8m. Disappointedly, incorpo-
ration of a 3′-methoxy group in the 4′-pyridinyl candidate
causes no change in potency. Given that the 3,4-dipropargyl
ester of the 3′,5′-dimethoxy analogue (7g) is much more potent
than the 3,4-diethyl ester counterpart (7c), we also prepared
the 3,4-dipropargyl ester analogue of 8k. Disappointingly,
instead of improving potency, it suffered a slight loss of activity
(data not included).
Our previous results and the results from heterocyclic
analogues suggest that the meta and para positions of the 6-
phenyl ring prefer hydrophilic functional groups, but the impact
of size had not been fully investigated. We therefore introduced
varied amino functional groups at these two positions to
confirm the preference of hydrophilicity and to explore the
optimal size (Table 4). Consistent with our observation in
series 7, the 3,4-dipropargyl esters (9c, 9g, 9j, 9k, 9m, and 9u)
are significantly more potent than the propargyl ethyl ester (9b,
9f, 9i, 9l, and 9t) and the diethyl ester (9a, 9e, and 9h) while an
introduction of a cyano functional group leads to a 40-fold loss
of activity (9d). Generally speaking, primary amino or
secondary amino functional groups are tolerated while tertiary
amino functional groups (9k and 9s) lead to a 4- to 12-fold loss
of activity. With respect to the secondary amino functional
group, the smaller methylamino substituent (9i, 9j, and 9r) is
preferred over the larger ethylamino substituent (9l, 9m, and
9u). The carboxyamido functional group (9p vs 9a) is
tolerated, while the chloroacetyl substitution (9o vs 9c) leads
to a 4-fold loss of activity. Replacement of the 3′-NH2 in 9c by
1H-tetrazole (9q) does not affect the cytotoxicity, whereas the
introduction of 2-hydroxyethyl group (9n) improves the
activity by 2-fold. These results overall confirmed that
Incorporating fluorine on the 6-phenyl ring of the 4H-
chromene does not show significant improvement over the
parent compound (sHA 14-1) except 8f (Table 2). Among the
Table 2. IC50 (μM) of Fluorine-Containing Analogues 8b−f
in HL60
a
compd
R1
R2
R3
R4
IC50 SEM
sHA 14-129
H
F
H
H
F
H
H
H
F
H
H
H
H
H
F
91.0 0.3
73.0 12.3
76.4 6.2
72.1 8.0
76.6 6.9
35.9 4.3
8b
8c
8d
8e
8f
H
H
F
H
H
F
F
H
H
a
Results are given as the mean of at least two independent
experiments with triplicates in each experiment.
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Table 3. IC50 (μM) of Heterocyclic Analogues 8g−m in
HL60
Table 4. IC50 (μM) of Analogues 9a−u in HL60
a
compd
R1
Et
R2
Et
R3
R4
IC50 SEM
9a
9b
9c
9d
9e
9f
NH2
NH2
NH2
H
H
H
20.2 2.1
3.4 0.3
C3H3
C3H3
Et
C3H3
1.24 0.27
43.9 4.0
22.3 1.3
10.5 0.4
0.61 0.08
7.45 0.17
7.13 0.29
0.88 0.06
Et
Et
H
NH2
NH2
NH2
H
C3H3
C3H3
Et
Et
H
9g
9h
9i
C3H3
Et
H
NHMe
NHMe
NHMe
NMe2
NHEt
NHEt
C3H3
C3H3
C3H3
C3H3
C3H3
C3H3
C3H3
Et
Et
H
9j
C3H3
C3H3
Et
H
9k
9l
H
12.00 0.71
17.2 3.49
1.86 0.45
0.63 0.28
5.3 0.35
H
9m
9n
9o
9p
9q
9r
9s
9t
C3H3
C3H3
C3H3
Et
H
NHCH2CH2OH
H
NHCOCH2Cl
H
CONH2
H
5.40 0.07
1.62 0.15
2.45 0.42
2.56 0.26
33.0 9.9
C3H3
C3H3
C3H3
C3H3
C3H3
C3H3
C3H3
C3H3
Et
tetrazole
H
H
H
H
H
NHMe
NMe2
NHEt
NHEt
9u
C3H3
7.27 1.23
a
Results are given as the mean of at least two independent
experiments with triplicates in each experiment.
CML, substantiating the clinical relevance of these MDR cancer
cell line models. These cell lines acquire MDR through
different mechanisms. For instance, the apoptotic machinery is
impaired in HL60/DOX via the up-regulation of c-inhibitor of
apoptosis (c-IAP).40 HL60/MX2 confers its resistance against
mitoxantrone via down-regulating of topoisomerase IIβ with no
involvement of p-glycoprotein.41,42 CCRF-CEM/C2 and
CCRF-CEM/VM-1-5 gain resistance via target mutation.43−45
HL60/MX2 and CCRF-CEM/C2 also have elevated levels of
antiapoptotic Bcl-2 family proteins.29 On the other hand,
HL60/ADR, HL60/DNR, K562/DOX, K562/HHT300, and
CCRF-CEM/VLB100 mediate their resistance at least partially
via the overexpression of p-glycoprotein.46,47 These parental
and MDR cell lines were evaluated for their sensitivity against a
panel of standard therapies in a dose-dependent manner to
determine the IC50 values. The ratio of the IC50 against MDR
cell line to the IC50 against corresponding parental cell line is
summarized in Table 5 with the IC50 for the parental lines
presented as well. As expected, the MDR cell lines generally
demonstrate cross-resistance to standard cancer therapies
(Table 5). Since several of these (HL60/MX2, HL60/ADR,
HL60/DNR, HL60/DOX, K562/DOX, and CCRF-CEM/VM-
1-5) acquire their resistance through exposure to topoisomerase
II inhibitors, they all demonstrate significant cross-resistance to
doxorubicin, mitoxantrone, and etoposide, three topoisomerase
II inhibitors. Similarly, K562/HHT300 demonstrates significant
cross-resistance to vincristine given that its resistance was
developed upon exposure to an antimicrotubule agent. On the
other hand, CCRF-CEM/C2 reveals no or weak cross-
a
Results are given as the mean of at least two independent
experiments with triplicates in each experiment.
hydrophilic functional groups at the meta and para positions of
the 6-phenyl are preferred. The size of the substitutions at the
para position needs to be small, while the meta position has
more tolerance on the size of the functional group. Among
these analogues, 9g, 9j, and 9n reveal submicromolar potency.
Characterization of Nine Pairs of Parental and MDR
Cancer Cell Lines (Table 5). In order to evaluate the scope of
our CXL candidate against MDR in cancer treatment, we
acquired four pairs of AML cell lines (HL60 vs HL60/MX2,
HL60/ADR, HL60/DNR, and HL60/DOX), three pairs of
ALL cell lines (CCRF-CEM vs CCRF-CEM/C2, CCRF-CEM/
VM-1-5, and CCRF-CEM/VLB100), and two pairs of CML
cancer lines (K562 vs K562/DOX and K562/HHT300). The
MDR cell lines were developed from the parental cell lines
through chronic exposure to different cancer therapies,
including mitoxantrone (a topoisomerase II inhibitor, MX2),
adriamycin (a topoisomerase II inhibitor, ADR, also named as
doxorubicin), daunorubicin (a topoisomerase II inhibitor,
DNR), doxorubicin (a topoisomerase II inhibitor, DOX),
camptothecin (a topoisomerase I inhibitor, C2), homoharring-
tonine (an antimicrotubule agent, HHT), and vinblastine (an
antimicrotubule agent, VLB). Most of these cancer therapies
are the first- or second-line therapies against AML, ALL, or
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a
Table 5. Relative Sensitivity of MDR Cancer Cell Lines against Standard Therapies
b
relative sensitivity with IC50 for the corresponding parental line in parentheses
resistant cell line
doxorubicin (μM)
vincristine (nM)
Ara-C (μM)
mitoxantrone (μM)
etoposide (μM)
HL60/MX2
5.3 (0.14)
>15 (0.32)
>50 (0.32)
>100 (0.04)
>10 (0.44)
>20 (0.44)
6.7 (0.46)
>15 (0.11)
6.1 (0.11)
1 (0.06)
0.4 (4.0)
0.1 (5.6)
1.3 (5.6)
7.5 (0.049)
1.5 (14)
>20 (0.016)
2.4 (0.97)
>10 (0.97)
>20 (3.1)
9.0 (1.6)
HL60/ADR
1 (0.1)
>10 (8.1)
>50 (8.1)
>200 (0.11)
>15 (2.3)
>1000 (2.3)
2.5 (0.24)
>50 (0.99)
8.6 (0.99)
HL60/DNR
>1000 (0.1)
>1000 (0.62)
>1000 (0.1)
>1000 (0.1)
1 (0.59)
HL60/DOX
K562/DOX
5.1 (0.51)
>10 (0.51)
3.0 (0.036)
>30 (0.085)
2.3 (0.085)
K562/HHT300
CCRF-CEM/C2
CCRF-CEM/VM-1-5
CCRF-CEM/VLB100
7.0 (14)
4.1 (0.006)
6.5 (0.02)
1 (0.02)
1 (0.17)
>1000 (0.17)
a
Relative sensitivity is defined as the ratio of IC50 in the resistant cell line relative to that in the corresponding parental cell line. When relative
sensitivity is >1, MDR cell shows resistance to the therapy. When relative sensitivity is <1, MDR cell shows collateral sensitivity to the therapy.
b
Results are given as the mean of at least two independent experiments with triplicates in each experiment. Because these cell lines were developed
from different sources as detailed in the Experimental Section, the parental cell lines of the same cell line do not demonstrate the same sensitivity to
the same therapy.
a
Table 6. Relative sensitivity of MDR cancer cell lines towards 1, 3, 6, and 9g
b
relative sensitivity with IC50 for the parental line in parentheses
resistant cell line
1 (μM)
3 (μM)
6 (μM)
9g (μM)
HL60/MX2
1.5 (1.39)
4.5 (10.5)
4.9 (10.5)
19.7 (0.76)
2.7 (34.7)
2.3 (34.7)
0.2 (0.74)
2.2 (1.17)
0.3 (1.17)
0.86 (19.8)
0.47 (30.6)
0.64 (30.6)
1.0 (6.8)
0.02 (3.0)
1.0 (0.078)
>50 (0.078)
>50 (0.007)
4.2 (0.17)
>10 (0.17)
1.45 (0.27)
1.0 (0.31)
7.7 (0.31)
1.0 (0.62)
0.26 (0.89)
0.27 (0.89)
0.60 (0.28)
1.08 (2.36)
1.1 (2.36)
0.54 (1.47)
0.42 (1.82)
0.93 (1.82)
HL60/ADR
HL60/DNR
HL60/DOX
K562/DOX
0.47 (26.9)
0.43 (26.9)
0.58 (7.05)
2.1 (13.7)
0.66 (13.7)
K562/HHT300
CCRF-CEM/C2
CCRF-CEM/VM-1-5
CCRF-CEM/VLB100
a
Relative sensitivity is defined as the ratio of the IC50 in the resistant cell line relative to that in the corresponding parental cell line. When relative
sensitivity is >1, MDR cell shows resistance to the therapy. When relative sensitivity is <1, MDR cell shows collateral sensitivity to the therapy.
b
Results are given as the mean of at least two independent experiments with triplicates in each experiment. Because these cell lines were developed
from sources as detailed in the Experimental Section, the parental cell lines of the same cell line do not demonstrate the same sensitivity to the same
therapy.
resistance toward the therapies evaluated. This is reasonable,
since its resistance was developed upon exposure to
camptothecin, a topoisomerase I inhibitor, via topoisomerase
I mutation.43,44 There are three additional interesting
observations. First, HL60/ADR and HL60/DOX both are
derived from HL60 upon exposure to the same topoisomerase
II inhibitor, doxorubicin. They, however, reveal quite distinct
drug sensitivity profiles in that HL60/DOX demonstrates a
greater extent of cross-resistance while HL60/ADR even shows
collateral sensitivity toward Ara-C. On the basis of reported
characterization, they acquire resistance through different
mechanisms. Such a distinction may be caused by multiple
factors, such as different treatment regimens when HL60 cells
were exposed to adriamycin/doxorubicin during the resistance
development period. Second, these resistant cell lines reveal
either substantial (>1000-fold) or no resistance to vincristine.
On the basis of the reported characterization of these cell lines,
HL60/DNR, K562/DOX, K560/HHT300, and CCRF-CEM/
VLB100 all overexpress p-glycoprotein while HL60/DOX has
not been well characterized for this protein. Since vincristine is
an excellent substrate for drug efflux, p-glycoprotein over-
expression is probably one major resistant mechanism among
these cell lines to vincristine. Third, none of these MDR cell
lines reveals >10-fold resistance to Ara-C. Indeed, two of them
reveal collateral sensitivity to Ara-C. These data suggest that
Ara-C is less likely to suffer cross-resistance derived from the
nonantimetabolite cancer therapies evaluated herein. Such an
observation is consistent with the reported major mechanism
responsible for Ara-C resistance: reduction in the activity of
deoxycytidine kinase (dCK), an enzyme in the rate limiting step
for Ara-C activation,48 which is unlikely to change in these
MDR cancer cells. These data overall demonstrate that there
are several different mechanisms responsible for MDR among
these cancer cells, establishing a system to evaluate the scope of
our lead compound (9g) against MDR in comparison to several
leads that target antiapoptotic Bcl-2 family proteins (1), p-
glycoprotein (3), or SERCA (6).
Sensitivity Profiling of the Nine Pairs of Parental and
MDR Cancer Cell Lines to 1, 3, 6, and 9g (Table 6). 1 is
selected for evaluation because it is one of the most potent
inhibitors against the antiapoptotic Bcl-2 family proteins,
although it does not inhibit Mcl-1.17 3 is a unique small
molecule developed by Gottesman et al. that selectively kills
cancer cell lines that overexpress p-glycoprotein.21 6 is a potent
inhibitor against SERCA and has been used as a lead for
prodrug development against prostate cancer.49 9g is selected
as the lead of our CXL candidates in this multi-MDR cell line
screening because of its submicromolar potency, being the most
potent candidate among the newly synthesized candidates.
These four compounds were evaluated for their cytotoxicity
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against the parental and MDR cell lines in a dose-dependent
manner to determine their IC50 values. The ratio of the IC50
against MDR cell line to the IC50 against the corresponding
parental cell line is summarized in Table 6. With respect to 1,
seven of the nine MDR cell lines reveal 2- to 20-fold resistance
and two reveal significant collateral sensitivity. These data
suggest that targeting the antiapoptotic Bcl-2 family proteins
alone, particularly targeting only a subset of the antiapoptotic
Bcl-2 family proteins, is not sufficient to overcome MDR. Since
c-IAP is downstream of the antiapoptotic Bcl-2 family proteins
along the mitochondrial apoptotic pathway, HL60/DOX
demonstrates the greatest extent of resistance to 1 (20-fold).
Consistent with the results reported by Gottesman et al. in
other MDR models, MDR cancer cells that overexpress p-
glycoprotein, such as HL60/ADR, HL60/DNR, K562/DOX,
K562/HHT300, and CCRF-CEM/VLB100, reveal collateral
sensitivity to 3. CCRF-CEM/C2 also shows decent collateral
sensitivity to 3, although its ability to accumulate camptothecin
has been reported to be similar as the parental cells,50
suggesting no change in the activity of ABC transporter
proteins. HL60/MX2, HL60/DOX, and CCRF-CEM/VM-1-5
fail to show collateral sensitivity to 3 given that they acquire
resistance via target mutation/reduction or impairment of
apoptotic machinery, independent of p-glycoprotein. Indeed,
CCRF-CEM/VM-1-5 reveals a 2-fold resistance to 3. These
results suggest that 3 is unlikely to be effective against p-
glycoprotein independent MDR. Six of the nine MDR cell lines
confer cross-resistance to 6, while HL60/MX2 demonstrates a
50-fold collateral sensitivity to 6. The mechanism of this
significant selectivity is under investigation. For 9g, five of the
nine MDR cell lines show collateral sensitivity, and none of
them demonstrate any resistance. Since these cells acquire
MDR through several mechanisms, our results support the
hypothesis that 9g targets several key mechanisms involved in
MDR and that cancer cells are unlikely to acquire resistance to
9g relative to 1, 3, and 6.
mechanisms (Table 5). These MDR cancer cell lines generally
reveal cross-resistance toward standard therapies. Among the
three classes of anticancer agents evaluated, topoisomerase II
inhibitors suffer the greatest extent of cross-resistance; all nine
MDR cancer cell lines are resistant to all three topoisomerase II
inhibitors. This is not surprising given that most of these MDR
cell lines acquire their resistance upon exposure to topoisomer-
ase inhibitors. Vincristine generally suffers either significant
resistance when the cells overexpress p-glycoprotein or no
resistance. All MDR cell lines show no or weak resistance to
Ara-C, consistent with its distinct mechanisms of action and
resistance.
Candidate 1 suffers cross-resistance from seven of the nine
MDR cancer cell lines, especially when the resistance is
acquired downstream of the mitochondrial Bcl-2 family
proteins (HL60/DOX). Candidate 3 suffers cross-resistance
from one MDR cell line, which does not overexpress p-
glycoprotein. Candidate 6 suffers cross-resistance from six of
these cell lines, four of which have been characterized to
overexpress p-glycoprotein. Candidate 9g, on the other hand,
suffers no cross-resistance from any of these nine MDR cancer
cell lines. Two MDR cell lines reveal collateral sensitivity to
candidate 1, and the mechanism remains to be established.
HL60/MX2 cell line shows a 50-fold collateral sensitivity to
candidate 6. Six of these MDR cell lines reveal collateral
sensitivity toward candidate 3, five of which have been
characterized to overexpress p-glycoprotein, consistent with
the observation from Dr. Gottesman et al.21 Five of these MDR
cell lines reveal collateral sensitivity toward candidate 9g, and
these five MDR cells have distinct mechanisms, including
overexpression of antiapoptotic Bcl-2 family proteins (CCRF-
CEM/C2),29 overexpression of p-glycoprotein (HL60/ADR
and HL60/DNR), apoptotic defect downstream of the
mitochondrial Bcl-2 family proteins (HL60/DOX), and target
mutation (CCRF-CEM/VM-1-5 and CCRF-CEM/C2). Most
importantly, each of these nine MDR cancer cell models
evaluated herein reveals collateral sensitivity to at least one
candidate among 1, 3, 6, and 9g.
CONCLUSION
■
In conclusion, we have performed SAR studies of CXL
candidates and defined the optimal functional groups at the 3,
4, and 6 positions of the 4H-chromene system, leading to the
identification of more potent candidates. One lead compound,
9g, demonstrates submicromolar potency against all the cancer
cell lines evaluated (Table 6), 5- to 35-fold more potent than 3
and can be >10 fold more potent than 1. 9g also reveals
selective anticancer potential toward MDR cancer cell models
that have different resistant mechanisms. 9g in combination
with 1, 3, and 6 represents a set of promising leads that may
help conquer MDR in cancer treatment.
On the basis of the potential of 4 to target MDR, we have
performed a detailed SAR study to characterize the optimal
functional groups at the 3, 4, and 6 positions of the 4H-
chromene system in order to improve the potency of the lead
candidate. The 3 and 4 positions are very sensitive to the size,
flexibility, and lipophilicity of the functional groups with
propargyl esters being the best (Tables 1 and 4). Increasing
flexibility or hydrophilicity significantly reduces the cytotoxicity
of the candidates. Similarly, increasing or significantly
decreasing the size of the functional groups also leads to a
decrease in cytotoxicity. At the 6 position, incorporation of
fluorophenyl or heterocyclic aryl systems has no or a limited
improvement on potency (Tables 2 and 3). The functional
groups at the meta and para positions on the 6-phenyl position,
however, can significantly affect the cytotoxicity of the
candidate (Table 4). Typically these positions prefer small
and hydrophilic functional groups. These findings culminate in
the discovery of 9g, 9j, and 9n with IC50 of 600−800 nM
against HL60 cells, which are about 20 times more potent than
4.
EXPERIMENTAL SECTION
■
Chemistry. All commercial reagents and anhydrous solvents were
purchased from vendors and were used without further purification or
distillation unless otherwise stated. Analytical thin layer chromatog-
raphy was performed on Whatman silica gel 60 Å with fluorescent
indicator (partisil K6F). Compounds were visualized by UV light and/
or stained with iodine or potassium permanganate solution followed
by heating. Flash column chromatography was performed on
Whatman silica gel 60 Å (230−400 mesh). NMR (1H and 13C)
spectra were recorded on a Varian 400 MHz or a Bruker 400 MHz
spectrometer and calibrated using an internal reference. ESI mode
mass spectra were recorded on a Bruker BiotofII mass spectrometer.
All the compounds synthesized are racemic mixtures. Purity of the
compounds was analyzed by HPLC using 75:25 ACN/H2O as the
We have also evaluated the scope of our lead candidate, 9g,
against MDR in cancer treatment and compared it with several
leads that each target specific mechanisms involved in MDR.
We first characterized the sensitivity of nine MDR cancer cell
lines, which acquire their drug resistance via different
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mobile phase with a flow rate of 0.9 mL/min on a C18 column. All
compounds exhibited greater than 95% purity.
derivatives were synthesized from their corresponding boronic acid
MIDA ester) were taken in toluene/water (10:2) (15 mL) and
degassed well with nitrogen. To this, Pd(PPh3)4 (0.05 mol %) was
added, and the mixture was stirred at 80−90 °C under an atmosphere
of nitrogen overnight. It was then acidified using HCl (1 N) on an ice
bath (for pyridyl derivatives, HCl wash was not given), extracted with
ethyl acetate (25 mL × 3), washed with water, brine, and dried over
MgSO4. Removal of the solvent under reduced pressure gave the crude
mass which upon purification by flash column chromatography gave
the desired 6-aryl-substituted coumarin.
6-(2,4-Difluorophenyl)-2H-chromen-2-one (11d). Base: so-
dium carbonate (4.5 mmol). Solvent: ethanol/water/toluene in 1:2:3
ratio (5 mL). Yield: 48%. 1H NMR (400 MHz, CDCl3): δ 7.75 (1H, d,
J = 9.6 Hz), 7.65 (1H, dd, J = 8.8, 1.2 Hz), 7.62 (1H, s), 7.45−7.35
(2H, m), 7.04−6.88 (2H, m), 6.47 (1H, d, J = 9.6 Hz).
6-(3,5-Difluorophenyl)-2H-chromen-2-one (11e). Base: so-
dium carbonate (4.5 mmol). Solvent: ethanol/water/toluene in 1:2:3
ratio (10 mL). Yield: 62%. 1H NMR (400 MHz, CDCl3): δ 7.76 (1H,
d, J = 9.6 Hz), 7.71 (1H, dd, J = 8.6, 2.1 Hz), 7.65 (1H, d, J = 2.1 Hz),
7.42 (1H, d, J = 8.6 Hz), 7.15−7.05 (2H, m), 6.83 (1H, tt, J = 8.8, 2.2
Hz), 6.50 (1H, d, J = 9.6 Hz). 13C NMR (100 MHz, CDCl3): δ 163.40
(dd, JC−F = 247.8 Hz, 12.7 Hz), 160.30, 154.04, 143.08, 142.63 (t, JC−F
= 9.3 Hz), 135.43 (t, JC−F = 2.7 Hz), 130.42, 126.12, 119.18, 117.62,
117.51, 109.97 (dd, JC−F = 18.6 Hz, 7.4 Hz), 103.08 (t, JC−F = 25.3
Hz).
General Procedure for the Synthesis of Salicylaldehyde
(10a−c).29 5-Bromosalicylaldehyde (1 mmol), arylboronic acid (1.2
mmol), palladium(II) acetate (0.05 mmol), potassium carbonate (3
mmol), and triphenylphosphine (0.05 mmol) were added to 4 mL of
DMF/H2O (1:1) mixture and degassed well with nitrogen. The
mixture was then heated to 60 °C under N2 atmosphere for 6 h. The
crude mixture was diluted with water and extracted with ethyl acetate
(25 mL × 3) and washed with brine. The combined organic phase was
dried over Na2SO4 followed by solvent evaporation, which gave a
crude product which was further purified by column chromatography
to yield the pure product.
2′-Fluoro-4-hydroxy-[1,1′-biphenyl]-3-carbaldehyde (10a).
Yield: 77%. 1H NMR (400 MHz, CDCl3): δ 11.10 (1H, s), 9.98
(1H, s), 7.82−7.72 (2H, m), 7.45 (1H, td, J = 7.8, 1.7 Hz), 7.41−7.33
(1H, m), 7.31−7.17 (2H, m), 7.11 (1H, d, J = 8.6 Hz).
3′-Fluoro-4-hydroxy-[1,1′-biphenyl]-3-carbaldehyde (10b).
Yield: 76%. 1H NMR (400 MHz, CDCl3): δ 11.04 (1H, s), 9.98
(1H, s), 7.80−7.71 (2H, m), 7.42 (1H, td, J = 7.9, 6.1 Hz), 7.36−7.30
(1H, m), 7.29−7.21 (1H, m), 7.12−7.01 (2H, m).
4′-Fluoro-4-hydroxy-[1,1′-biphenyl]-3-carbaldehyde (10c).
Yield: 71%. 1H NMR (400 MHz, CDCl3): δ 11.00 (1H, s), 9.97
(1H, s), 7.74−7.66 (2H, m), 7.55−7.46 (2H, m), 7.18−7.10 (2H, m),
7.10−7.04 (1H, m).
General Procedure for the Synthesis of Coumarin (11a−c).29
To N,N-dimethylacetamide (1.98 mmol) stirred at 0 °C, phosphorus
oxychloride (1.98 mmol) was added slowly. The reaction mixture was
allowed to stir at 0 °C for 30 min followed by addition of the
corresponding salicylaldehyde (0.99 mmol) in dry DCM. The reaction
mass was then refluxed (at 60 °C) for about 3 h. Following this, it was
cooled to room temperature and saturated NaHCO3 solution (10 mL)
was added to it (solid formation was noticed). The combined mass
was heated again at 68−70 °C for another 30 min (the formed solid
dissolves and gets into DCM layer), cooled, and acidified (1 N HCl),
followed by extraction with methylene chloride. The extracts were
combined, dried (anhydrous MgSO4), and concentrated under
reduced pressure to afford a crude product, which upon base
treatment (NaOH) followed by recrystallization gave pure compound.
In some cases, purification was carried out by flash column
chromatography.
6-(Pyridin-2-yl)-2H-chromen-2-one (11f). Solvent: DMF/IPA
in 4:1 ratio (10 mL). Additive: Cu(OAc)2 (0.5 mmol). Yield: 61%. 1H
NMR (400 MHz, CDCl3): δ 8.73−8.66 (1H, m), 8.18 (1H, d, J = 2.2
Hz), 8.14 (1H, d, J = 8.8, 2.2 Hz), 7.84−7.71 (3H, m), 7.42 (1H, d, J =
8.8 Hz), 7.28 (1H, ddd, J = 7.2, 4.8, 1.3 Hz), 6.46 (1H, d, J = 9.6 Hz).
13C NMR (100 MHz, CDCl3): δ 160.50, 155.38, 154.41, 149.78,
143.52, 136.98, 135.76, 130.12, 126.25, 122.51, 120.16, 118.98, 117.12,
116.98.
6-(Pyridin-3-yl)-2H-chromen-2-one (11g). Solvent: dioxane/
water in 5:1 ratio (10 mL). Yield: 50%. 1H NMR (400 MHz, CDCl3):
δ 8.81 (1H, s), 8.59 (1H, d, J = 4.5 Hz), 7.84 (1H, d, J = 7.8 Hz), 7.76
(1H, d, J = 9.6 Hz), 7.69 (1H, d, J = 8.6 Hz), 7.65 (1H, s), 7.44−7.31
(2H, m), 6.44 (1H, d, J = 9.6 Hz).
6-(Pyridin-4-yl)-2H-chromen-2-one (11h). Solvent: dioxane/
water in 5:1 ratio (10 mL). Yield: 61%. 1H NMR (400 MHz, CDCl3):
δ 8.67 (2H, d, J = 6.0 Hz), 7.83−7.70 (3H, m), 7.48 (2H, d, J = 6.0
Hz), 7.41 (1H, d, J = 8.6 Hz), 6.47 (1H, d, J = 9.4 Hz). 13C NMR (100
MHz, CDCl3): δ 163.19, 160.23, 154.47, 150.53, 146.53, 143.10,
134.69, 130.40, 126.29, 121.46, 119.32, 117.78.
6-(2-Methoxypyridin-4-yl)-2H-chromen-2-one (11i). Solvent:
dioxane/water in 5:1 ratio (10 mL). Yield: 22%. 1H NMR (400 MHz,
CDCl3): δ 8.24 (1H, d, J = 5.5 Hz), 7.80−7.73 (2H, m), 7.71 (1H, d, J
= 2.2 Hz), 7.42 (1H, d, J = 8.6 Hz), 7.09 (1H, dd, J = 5.4, 1.1 Hz), 6.93
(1H, d, J = 1.1 Hz), 6.49 (1H, d, J = 9.6 Hz), 3.99 (3H, s). 13C NMR
(100 MHz, CDCl3): δ 164.97, 160.28, 154.36, 149.28, 147.58, 143.13,
134.81, 130.38, 126.20, 119.17, 117.61, 117.42, 115.02, 108.44, 53.62.
6-(Pyrimidin-5-yl)-2H-chromen-2-one (11j). Solvent: dioxane/
water in 5:1 ratio (10 mL). Yield: 12%. 1H NMR (400 MHz, CDCl3):
δ 9.25 (1H, s), 8.97 (2H, s), 7.79 (1H, d, J = 9.6 Hz), 7.74 (1H, dd, J =
8.6, 2.2 Hz), 7.69 (1H, d, J = 2.2 Hz), 7.50 (1H, d, J = 8.6 Hz), 6.53
(1H, d, J = 9.6 Hz). 13C NMR (100 MHz, CDCl3): δ 160.01, 157.92,
154.81, 154.43, 142.77, 132.86, 130.82, 130.30, 126.20, 119.60, 118.21,
117.89.
1
6-(2-Fluorophenyl)-2H-chromen-2-one (11a). Yield: 46%. H
NMR (400 MHz, CDCl3): δ 7.75 (1H, d, J = 9.6 Hz), 7.70 (1H, d, J =
8.6 Hz), 7.66 (1H, s), 7.48−7.31 (3H, m), 7.24 (1H, t, J = 7.2 Hz),
7.18 (1H, dd, J = 10.5, 8.5 Hz), 6.46 (1H, d, J = 9.6 Hz). 13C NMR
(100 MHz, CDCl3): δ 160.58, 159.62 (d, JC−F = 247.0 Hz), 153.44,
143.37, 132.46 (d, JC−F = 2.2 Hz), 132.19, 130.47 (d, JC−F = 3.7 Hz),
129.60 (d, JC−F = 8.2 Hz), 128.15 (d, JC−F = 3.7 Hz), 127.18 (d, JC−F
=
13.5 Hz), 124.60 (d, JC−F = 3.7 Hz), 118.79, 116.99, 116.97, 116.25 (d,
JC−F = 23.1 Hz).
1
6-(3-Fluorophenyl)-2H-chromen-2-one (11b). Yield: 48%. H
NMR (400 MHz, CDCl3): δ 7.76 (1H, d, J = 9.6 Hz), 7.72 (1H, dd, J
= 8.7, 1.9 Hz), 7.66 (1H, d, J = 2.2 Hz), 7.47−7.38 (3H, m), 7.31−
7.24 (1H, m), 7.08 (1H, td, J = 8.3, 2.5 Hz), 6.48 (1H, d, J = 9.6 Hz).
13C NMR (100 MHz, CDCl3): δ 163.19 (d, JC−F = 245.5 Hz), 160.47,
153.69, 143.26, 141.57 (d, JC−F = 7.5 Hz), 136.47 (d, JC−F = 2.2 Hz),
130.57, 130.53 (d, JC−F = 8.2 Hz), 126.08, 122.65 (d, JC−F = 3.0 Hz),
119.08, 117.42, 117.26, 114.60 (d, JC−F = 20.9 Hz), 113.96 (d, JC−F
=
3-(2-Oxo-2H-chromen-6-yl)benzamide (11k). Solvent: diox-
22.2 Hz).
1
1
ane/water in 5:1 ratio (10 mL). Yield: 13.7%. H NMR (400 MHz,
6-(4-Fluorophenyl)-2H-chromen-2-one (11c). Yield: 49%. H
NMR (400 MHz, CDCl3): δ 7.75 (1H, d, J = 9.4 Hz), 7.69 (1H, d, J =
8.5 Hz), 7.62 (1H, d, J = 2.1 Hz), 7.58−7.47 (2H, m), 7.38 (1H, dd, J
= 8.5, 2.1 Hz), 7.15 (2H, t, J = 8.3 Hz), 6.46 (1H, d, J = 9.4 Hz). 13C
NMR (100 MHz, CDCl3): δ 162.64 (d, JC−F = 245.5 Hz), 160.55,
153.33, 143.30, 136.80, 135.50 (d, JC−F = 3.0 Hz), 130.53, 128.62 (d,
JC−F = 8.2 Hz), 125.88, 119.02, 117.27, 117.13, 115.89 (d, JC−F = 21.6
Hz).
CDCl3): δ 8.22 (1H, t, J = 1.9 Hz), 8.18−8.07 (3H, m), 8.00 (1H, dd,
J = 8.6, 2.3 Hz), 7.93−7.84 (2H, m), 7.58 (1H, t, J = 7.6 Hz), 7.53
(1H, d, J = 8.6 Hz), 7.47 (1H, br s), 6.57 (1H, d, J = 9.6 Hz). 13C
NMR (100 MHz, CDCl3): δ 168.08, 160.34, 153.60, 144.72, 139.03,
136.29, 135.50, 130.84, 129.81, 129.51, 127.25, 126.97, 126.15, 119.61,
117.37, 117.15.
6-(3-Nitrophenyl)-2H-chromen-2-one (11l). Yield: 61%. 1H
NMR (400 MHz, CDCl3): δ 8.46 (1H, t, J = 2.0 Hz), 8.26−8.24 (1H,
m), 7.93−7.91 (1H, m), 7.81−7.74 (3H, m), 7.66 (1H, t, J = 8 Hz),
7.47 (1H, d, J = 8.8 Hz), 6.52 (1H, d, J = 9.6 Hz). 13C NMR (100
General Procedure for the Synthesis of 6-Aryl-Substituted
Coumarin (11d−o). 6-Bromocoumarin (1 mmol), K2CO3 (2.5
mmol), and the respective boronic acid (1.2 mmol; all the heterocyclic
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MHz, CDCl3): δ 160.40, 154.29, 148.98, 143.18, 141.21, 135.36,
133.03, 130.71, 130.22, 126.49, 122.69, 122.04, 119.49, 117.97, 117.81.
6-(4-Nitrophenyl)-2H-chromen-2-one (11m). Yield: 58%. 1H
NMR (400 MHz, CDCl3): δ 8.34 (2H, d, J = 8.8 Hz), 7.81−7.73 (5H,
m), 7.47 (1H, d, J = 8.4 Hz), 6.52 (1H, d, J = 9.6 Hz). 13C NMR (100
MHz, CDCl3): δ 160.37, 154.48, 147.52, 145.83, 143.19, 135.45,
130.89, 127.93, 126.74, 124.48, 119.47, 117.95, 117.79.
CDCl3): δ 166.77, 156.94, 154.94, 144.36, 141.29, 139.19, 132.65,
129.73, 129.06, 128.99, 124.33, 124.11, 124.02, 123.96, 120.05, 110.73,
80.43, 55.79, 51.47, 37.54, 28.52.
tert-Butyl Ethyl-(3-(2-oxo-2H-chromen-6-yl)phenyl)-
carbamate (11s). Yield: 80%. 1H NMR (400 MHz, CDCl3): δ
7.76 (1H, d, J = 9.6 Hz), 7.71 (1H, dd, J = 2.4, 8.8 Hz), 7.65 (1H, d, J
= 2.0 Hz), 7.42−7.37 (4H, m), 7.23−7.20 (1H, m), 6.45 (1H, d, J =
9.6 Hz), 3.72 (2H, q, J = 7.2 Hz), 1.46 (9H, s), 1.19 (3H, t, J = 7.0
Hz). 13C NMR (100 MHz, CDCl3): δ 160.73, 154.57, 153.61, 148.54,
143.33, 140.16, 137.43, 130.83, 129.44, 126.42, 126.21, 125.83, 124.66,
119.14, 117.37, 117.19, 80.32, 45.19, 28.50, 14.12.
tert-Butyl (3-(2-Oxo-2H-chromen-6-yl)phenyl)carbamate
1
(11n). Yield: 75%. H NMR (400 MHz, CDCl3): δ 7.78−7.74 (3H,
m), 7.68 (1H, d, J = 2.0 Hz), 7.39−7.35 (2H, m), 7.23 (2H, dd, J = 2.2,
8 Hz), 6.59 (1H, s), 6.46 (1H, d, J = 9.56 Hz), 1.54 (9H, s). 13C NMR
(100 MHz, CDCl3): δ 160.87, 153.66, 152.85, 143.64, 140.52, 139.22,
137.72, 130.98, 129.71, 126.30, 121.85, 119.14, 117.86, 117.37, 117.18,
80.94, 28.49 (two coinciding carbon resonances).
tert-Butyl Methyl-(4-(2-oxo-2H-chromen-6-yl)phenyl)-
1
carbamate (11t). Yield: 78%. H NMR (400 MHz, CDCl3): δ 7.76
(1H, d, J = 9.6 Hz), 7.73 (1H, dd, J = 2, 8.8 Hz), 7.64 (1H, d, J = 2.0
Hz), 7.53 (2H, d, J = 8.4 Hz), 7.40−7.34 (3H, s), 6.46 (1H, d, J = 9.6
Hz), 3.31 (3H, s), 1.49 (9H, s). 13C NMR (100 MHz, CDCl3): δ
160.65, 154.62, 153.35, 143.43, 137.18, 136.15, 130.57, 127.51, 127.10,
125.84, 125.72, 119.03, 117.27, 117.06, 80.60, 37.19, 28.34.
tert-Butyl (4-(2-Oxo-2H-chromen-6-yl)phenyl)carbamate
1
(11o). Yield: 78%. H NMR (400 MHz, CDCl3): δ 7.75 (1H, d, J =
9.6 Hz), 7.71 (1H, dd, J = 2, 8.4 Hz), 7.62 (1H, d, J = 2.0 Hz), 7.52−
7.46 (4H, m), 7.37 (1H, d, J = 8.4 Hz), 6.59 (1H, s), 6.45 (1H, d, J =
9.6 Hz), 1.54 (9H, s). 13C NMR (100 MHz, CDCl3): δ 160.89, 153.33,
152.79, 143.65, 138.37, 137.43, 134.09, 130.53, 127.68, 125.69, 119.17,
119.05, 117.37, 117.14, 80.96, 28.48.
tert-Butyl Ethyl-(4-(2-oxo-2H-chromen-6-yl)phenyl)-
carbamate (11u). Yield: 81%. 1H NMR (400 MHz, CDCl3): δ
7.75 (1H, d, J = 9.6 Hz), 7.71 (1H, dd, J = 2.4, 8.8 Hz), 7.64 (1H, d, J
= 2.0 Hz), 7.52 (2H, d, J = 8.4 Hz), 7.37 (1H, d, J = 8.4 Hz), 7.29 (2H,
d, J = 8.4 Hz), 6.44 (1H, d, J = 9.6 Hz), 3.71 (2H, q, J = 7.2 Hz), 1.46
(9H, s), 1.18 (3H, t, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3): δ
160.74, 154.52, 153.47, 143.56, 142.37, 137.27, 136.82, 130.68, 127.42,
127.36, 126.00, 119.14, 117.34, 117.13, 80.35, 45.05, 28.46, 14.05.
General Procedure for Deprotection of Boc-amine by TFA
(11v−za). To a stirred solution of the respective Boc-amine (1 mmol)
in dry DCM, TFA (5 mmol) was added slowly at low temperature
under nitrogen. Stirring was continued for about 6 h, and then the
reaction was quenched with 10% bicarbonate solution. The mixture
was extracted with DCM, washed with water, brine, and dried over
MgSO4. The crude product obtained after removal of solvent itself was
pure enough to use for further reaction.
General Procedure for Reduction of Nitro to Amine (11p
and 11q). To a stirred suspension of the respective nitrocoumarin 11l
or 11m (5 mmol) in methanol (20 mL), 10% Pd/C (0.4 g) was added
followed by dry ammonium formate (25−30 mmol). The contents
were stirred well under nitrogen at room temperature. Upon
completion of reaction, the catalyst was removed by filtration through
Celite bed and the solvent was evaporated under reduced pressure.
The resulting mass was extracted with ethyl acetate, washed with
water, and dried (over MgSO4), and the solvent was removed under
reduced pressure. The crude solid was purified by flash chromatog-
raphy to isolate the desired amino derivative.
1
6-(3-Aminophenyl)-2H-chromen-2-one (11p). Yield: 65%. H
NMR (400 MHz, CDCl3): δ 7.76−7.71 (2H, m), 7.63 (1H, d, J = 2.4
Hz), 7.38 (1H, d, J = 8.4 Hz), 7.26−7.23 (1H, m), 6.96 (1H, d, J = 8
Hz), 6.88 (1H, t, J = 1.8 Hz), 6.71 (1H, dd, J = 1.6, 8 Hz), 6.46 (1H, d,
J = 9.2 Hz), 3.79 (2H, s). 13C NMR (100 MHz, CDCl3): δ 160.90,
153.51, 147.13, 143.67, 140.71, 138.18, 130.86, 130.10, 126.12, 119.06,
117.54, 117.25, 117.08, 114.64, 113.73.
6-(3-(Methylamino)phenyl)-2H-chromen-2-one (11v). Yield:
1
83%. H NMR (400 MHz, CDCl3): δ 7.75−7.71 (2H, m), 7.64 (1H,
d, J = 2.0 Hz), 7.36 (1H, d, J = 8.4 Hz), 7.27 (1H, t, J = 8 Hz), 6.90−
6.88 (1H, m), 6.76 (1H, t, J = 2 Hz), 6.64 (1H, dd, J = 2.0, 8.0 Hz),
6.44 (1H, d, J = 9.6 Hz), 3.88 (1H, s), 2.89 (3H, s). 13C NMR (100
MHz, CDCl3): δ 160.92, 153.43, 149.95, 143.71, 140.59, 138.56,
130.93, 129.94, 126.14, 119.01, 117.15, 116.97, 116.21, 112.04, 110.90,
30.85.
1
6-(4-Aminophenyl)-2H-chromen-2-one (11q). Yield: 72%. H
NMR (400 MHz, CDCl3): δ 7.74 (1H, d, J = 9.6 Hz), 7.68 (1H, dd, J
= 2, 8.8 Hz), 7.58 (1H, d, J = 2 Hz), 7.39 (2H, d, J = 8.4 Hz), 7.35
(1H, d, J = 8.4 Hz), 6.77 (2H, d, J = 8.4 Hz), 6.44 (1H, d, J = 9.6 Hz),
3.79 (2H, s). 13C NMR (100 MHz, CDCl3): δ 161.05, 152.92, 146.47,
143.76, 138.01, 130.24, 129.69, 128.08, 125.16, 119.13, 117.24, 116.96,
115.59.
Synthesis of N-Alkyl-Boc-amines (11r−u). To a stirred solution
of the respective amine (1 mmol) and NaH (1.5 mmol) in dry DMF
under nitrogen, the desired alkyl iodide (2.5 mmol) was added. The
contents were stirred well at room temperature. Upon completion,
water was added and the contents were extracted with DCM, washed
with water, cold brine, and dried over MgSO4. The crude mass
obtained by evaporation under reduced pressure was purified by flash
chromatography to afford the desired N-alkylated amines. When NaH
and alkyl iodide were used at 2.5 and 5 equiv, respectively, N-
alkylation followed by the lactone ring cleavage occurred leading to the
formation of 11r1 as the sole product.
6-(3-(Ethylamino)phenyl)-2H-chromen-2-one (11w). Yield
85%. 1H NMR (400 MHz, CDCl3): δ 7.77−7.72 (2H, m), 7.65
(1H, d, J = 2.4 Hz), 7.38 (1H, d, J = 8.4 Hz), 7.26 (1H, t, J = 7.8 Hz),
6.9 (1H, d, J = 7.6 Hz), 6.77 (1H, t, J = 2 Hz), 6.63 (1H, dd, J = 2.2,
8.4 Hz), 6.46 (1H, d, J = 9.2 Hz), 3.7 (1H, s), 3.23 (2H, q, J = 7.2 Hz),
1.29 (3H, t, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3): δ 160.96,
153.51, 149.11, 143.72, 140.71, 138.66, 130.99, 130.03, 126.18, 119.07,
117.24, 117.07, 116.27, 112.38, 111.34, 38.65, 15.04.
6-(4-(Methylamino)phenyl)-2H-chromen-2-one (11x). Yield:
1
85%. H NMR (400 MHz, CDCl3): δ 7.74 (1H, d, J = 9.6 Hz), 7.70
(1H, dd, J = 2.4, 8.8 Hz), 7.59 (1H, d, J = 2.4 Hz), 7.43 (2H, d, J = 8.8
Hz), 7.35 (1H, d, J = 8.8 Hz), 6.70 (2H, d, J = 8.8 Hz), 6.44 (1H, d, J
= 9.6 Hz), 3.89 (1H, br s), 2.89 (3H, s). 13C NMR (100 MHz,
CDCl3): δ 161.10, 152.79, 149.26, 143.82, 138.18, 130.16, 128.24,
127.98, 124.97, 119.14, 117.22, 116.91, 112.87, 30.81.
6-(4-(Ethylamino)phenyl)-2H-chromen-2-one (11y). Yield:
89%. 1H NMR (400 MHz, CDCl3): δ 7.73 (1H, d, J = 9.6 Hz),
7.68 (1H, dd, J = 2.4, 8.8 Hz), 7.58 (1H, d, J = 2 Hz), 7.41 (2H, d, J =
8.4 Hz), 7.33 (1H, t, J = 8.4 Hz), 6.68 (2H, d, J = 8.8 Hz), 6.44 (1H, d,
J = 9.6 Hz), 3.72 (1H, br s), 3.21 (2H, q, J = 7.2 Hz), 1.29 (3H, t, J =
7.6 Hz). 13C NMR (100 MHz, CDCl3): δ 161.09, 152.75, 148.36,
143.82, 138.16, 130.12, 128.12, 127.97, 124.92, 119.12, 117.19, 116.87,
113.15, 38.53, 14.96.
tert-Butyl Methyl(3-(2-oxo-2H-chromen-6-yl)phenyl)-
carbamate (11r). Yield: 72%. 1H NMR (400 MHz, CDCl3): δ
7.78−7.72 (2H, m), 7.66 (1H, d, J = 2.0 Hz), 7.48−7.36 (4H, m),
7.28−7.28 (1H, m), 6.47 (1H, d, J = 9.6 Hz), 3.32 (3H, s), 1.49 (9H,
s). 13C NMR (100 MHz, CDCl3): δ 160.77, 154.81, 153.65, 144.65,
143.55, 140.07, 137.54, 130.88, 129.34, 126.25, 124.83, 124.38, 124.14,
119.17, 117.42, 117.25, 80.68, 37.52, 28.52.
(cis)-Methyl 3-(3′-((tert-Butoxycarbonyl)(methyl)amino)-4-
methoxy-[1,1′-biphenyl]-3-yl)acrylate (11r1). Yield: 60% (12%).
1H NMR (400 MHz, CDCl3): δ 7.81 (1H, d, J = 2 Hz), 7.53 (1H, dd, J
6-(3-(Dimethylamino)phenyl)-2H-chromen-2-one (11z).
1
Yield: 65%. H NMR (400 MHz, CDCl3): δ 7.76 (1H, d, J = 9.5
= 2.0, 8.4 Hz), 7.42 (1H, s), 7.35 (2H, d, J = 4.8 Hz), 7.22−7.17 (2H,
m), 6.94 (1H, d, J = 8.8 Hz), 6.03 (1H, d, J = 12.4 Hz), 3.86 (3H, s),
3.68 (3H, s), 3.30 (3H, s), 1.47 (9H, s). 13C NMR (100 MHz,
Hz), 7.72 (1H, dd, J = 2, 8.2 Hz), 7.68 (1H, d, J = 2.4 Hz), 7.40 (1H,
d, J = 8.8 Hz), 7.33 (1H, t, J = 8.6 Hz), 6.92−6.88 (2H, m), 6.78
(1H,m), 6.46 (1H, d, J = 8.6 Hz), 3.01 (6H, s). 13C NMR (100 MHz,
5575
dx.doi.org/10.1021/jm300515q | J. Med. Chem. 2012, 55, 5566−5581

Journal of Medicinal Chemistry
Article
CDCl3): δ 160.82, 153.34, 151.03, 143.59, 140.44, 138.89, 130.99,
129.69, 126.15, 118.91, 117.07, 116.89, 115.48, 111.96, 111.13, 40.64.
6-(4-(Dimethylamino)phenyl)-2H-chromen-2-one (11za).
CDCl3): δ 7.52 (1H, dd, J = 2.25, 8.51 Hz), 7.40 (1H, d, J = 2.15 Hz),
7.29 (1H, d, J = 8.41 Hz), 6.64 (2H, d, J = 2.15 Hz), 6.48 (1H, t, J =
2.15 Hz), 5.76−5.95 (1H, m), 5.23−5.34 (2H, m), 5.19−5.26 (2H,
m), 5.12 (2H, dd, J = 4.60, 9.88 Hz), 5.04 (2H, br s), 4.64 (2H, tt, J =
1.20 Hz, J = 5.84 Hz), 4.59 (2H, tt, J = 1.20 Hz, 5.80 Hz), 3.85 (6H,
s), 3.29 (1H, dd, J = 9.98, 17.22 Hz), 2.74 (1H, dd, J = 4.50, 17.22
Hz). 13C NMR (100 MHz, CDCl3): δ 191.4, 171.1, 161.1, 160.0,
154.0, 148.1, 141.9, 139.6, 131.8, 130.6, 128.5, 128.0, 123.6, 119.7,
118.5, 105.5, 99.5, 66.9, 65.7, 55.4, 45.0, 35.8 (two coinciding carbon
resonances). MS (ESI, positive) m/z calcd for C26H27NO9 (M + H):
498.18; found, 498.16. HPLC purity: 95.6%.
1
Yield: 71%. H NMR (400 MHz, CDCl3): δ 7.74 (1H, d, J = 9.6
Hz), 7.7 (1H, dd, J = 2, 8.4 Hz), 7.6 (1H, d, J = 2.4 Hz), 7.48 (2H, d, J
= 8.8 Hz), 7.35 (1H, d, J = 8.8 Hz), 6.8 (2H, d, J = 8.4 Hz), 6.44 (1H,
d, J = 9.6 Hz), 3.01 (6H, s). 13C NMR (100 MHz, CDCl3): δ 161.09,
152.75, 150.34, 143.83, 138.07, 130.12, 127.74, 127.23, 124.91, 119.14,
117.21, 116.87, 112.88, 40.61.
General Procedure for the Synthesis of Substituted Alkyl-
4H-chromene-3-carboxylate Compounds (7a, 7b, 7j−l, 8a−m,
and 9a−u).29 Freshly cut sodium (0.096 mmol) was added to the
respective dry alcohol (5 mL), followed by the addition of ethyl
cyanoacetate (0.192 mmol). The reaction mixture was stirred at room
temperature under an inert atmosphere for 30 min, followed by the
addition of a solution of the corresponding coumarin (0.08 mmol) in
anhydrous alcohol or DCM (3 mL). The resulting reaction mixture
was stirred at room temperature. Upon consumption of the coumarin,
the reaction mass was concentrated, diluted with water (30 mL), and
extracted using ethyl acetate (3 × 20 mL). The organics were
combined, washed with water, brine, and dried (MgSO4). Removal of
the solvent under reduced pressure afforded the crude mass which
upon column purification gave the pure product. Note: Propargyl
alcohol used for this reaction must be previously dried with 3 Å
molecular sieves. The monopropargyl (Rf = 0.43) and dipropargyl (Rf
= 0.41) esters resulting from this reaction could be separated by flash
column using the acetone/hexane solvent (3:7) system.
Methyl 2-Amino-6-(3,5-dimethoxyphenyl)-4-(2-methoxy-2-
oxoethyl)-4H-chromene-3-carboxylate (7a). This compound
was synthesized by following the same general procedure except that
methyl cyanoacetate was used instead of ethyl cyanoacetate. Yield:
35%. 1H NMR (400 MHz, CDCl3): δ 7.45 (1H, d, J = 2.20 Hz), 7.41
(1H, dd, J = 2.25, 8.31 Hz), 7.03 (1H, d, J = 8.30 Hz), 6.67 (2H, d, J =
2.35 Hz), 6.46 (1H, t, J = 2.15 Hz), 6.35 (2H, br s), 4.35 (1H, dd, J =
4.70, 7.24 Hz), 3.85 (6H, s), 3.78 (3H, s), 3.59 (3H, s), 2.69 (1H, dd, J
= 4.70, 14.80 Hz), 2.62 (1H, dd, J = 7.20, 14.80 Hz). 13C NMR (100
MHz, CDCl3): δ 172.0, 169.4, 161.6, 161.1, 142.5, 137.5, 127.0, 126.5,
125.8, 116.1, 105.2, 105.0, 99.2, 76.5, 55.4, 51.4, 51.0, 43.5, 31.4. MS
(ESI, positive) m/z calcd for C22H24NO7 (M + H): 414.16; found,
414.21. HPLC purity: 100%.
Ethyl 2-(2-Amino-3-cyano-6-(3,5-dimethoxyphenyl)-4H-
chromen-4-yl)acetate (7k). This compound was synthesized by
following the same general procedure except that malononitrile was
1
used instead of ethyl cyanoacetate. Yield: 85%. H NMR (400 MHz,
CDCl3): δ 7.43−7.38 (2H, m), 7.00 (1H, d, J = 8.0 Hz), 6.64 (2H, d, J
= 2.0 Hz), 6.45 (1H, t, J = 2.0 Hz), 4.73 (2H, s), 4.14−4.09 (3H, m),
3.84 (6H, s), 2.74−2.72 (2H, m), 1.20 (3H, t, J = 6.8 Hz). 13C NMR
(100 MHz, CDCl3): δ 170.84, 161.26, 160.93, 148.97, 142.17, 138.29,
127.23, 126.78, 122.84, 119.76, 116.76, 105.37, 99.45, 60.87, 57.99,
55.56, 43.51, 32.14, 14.19. MS (ESI, positive) m/z calcd for
C22H22N2O5 (M + H): 395.15; found 395.2. HPLC purity: 100%.
Ethyl 2-(2-Amino-6-(3,5-dimethoxyphenyl)-3-(3-(4-methox-
yphenyl)-1,2,4-oxadiazol-5-yl)-4H-chromen-4-yl)acetate (7l).
To a solution of 4-methoxyphenylhydroximinoyl chloride (1 mmol),
and ethyl 2-(2-amino-3-cyano-6-(3,5-dimethoxyphenyl)-4H-chromen-
4-yl)acetate (1 mmol) in benzene was added triethylamine (1 mmol)
in benzene slowly. The contents were refluxed for 6 h and
concentrated under reduced pressure. This was diluted with water
and extracted with ethyl acetate. The organics were combined, washed
with water, brine, and dried over MgSO4. Removal of the solvent
under reduced pressure gave a crude mass which upon column
1
purification furnished pure product. Yield: 46%. H NMR (400 MHz,
CDCl3): δ 8.02 (2H, d, J = 8.8 Hz), 7.53 (1H, d, J = 2.0 Hz), 7.43 (1H,
dd, J = 2.0, 8.4 Hz), 7.07 (1H, d, J = 8.4 Hz), 6.98 (2H, d, J = 8.4 Hz),
6.69 (2H, d, J = 2 Hz), 6.59 (2H, br s), 6.47 (1H, t, J = 2 Hz), 4.57
(1H, t, J = 5.4 Hz), 4.02 (2H, q, J = 6.8 Hz), 3.87 (6H, s), 3.85 (3H,
s), 2.81 (2H, d, J = 5.6 Hz), 1.12 (3H, t, J = 6.8 Hz). 13C NMR (100
MHz, CDCl3): δ 175.63, 171.28, 166.96, 161.80, 161.26, 158.43,
149.69, 142.49, 137.95, 129.09, 127.25, 126.93, 124.60, 120.02, 116.45,
114.23, 105.38, 99.44, 72.25, 60.50, 55.57, 55.49, 43.10, 31.48, 14.19.
MS (ESI, positive) m/z calcd for C29H24N4O5 (M + H): 543.20; found
544.1. HPLC purity: 96.9%.
Ethyl 2-Amino-6-bromo-4-(2-ethoxy-2-oxoethyl)-4H-chro-
mene-3-carboxylate (8a). Yield: 85.0%. 1H NMR (400 MHz,
CDCl3): δ 7.38 (1H, d, J = 2.3 Hz), 7.28 (1H, dd, J = 8.6, 2.3 Hz),
6.84 (1H, d, J = 8.6 Hz), 6.30 (2H, br s), 4.28−4.17 (3H, m), 4.11−
3.99 (2H, m), 2.63 (1H, dd, J = 15.2, 4.5 Hz), 2.57 (1H, dd, J = 15.2,
4.5 Hz), 1.31 (3H, t, J = 7.0 Hz), 1.17 (3H, t, J = 7.1 Hz). 13C NMR
(100 MHz, CDCl3): δ 171.39, 168.73, 161.11, 149.03, 131.11, 130.55,
127.77, 117.50, 116.61, 76.23, 60.31, 59.58, 43.32, 31.06, 14.52, 14.08.
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(2-fluorophenyl)-
4H-chromene-3-carboxylate (8b). Yield: 18%. 1H NMR (400
MHz, CDCl3): δ 7.44 (1H, s), 7.42−7.34 (2H, m), 7.34−7.24 (1H,
m), 7.22−7.09 (2H, m), 7.01 (1H, d, J = 8.4 Hz), 6.34 (2H, br s), 4.35
(1H, dd, J = 6.7, 4.1 Hz), 4.24 (2H, q, J = 7.0 Hz), 4.02 (2H, q, J = 7.0
Hz), 2.75−2.55 (2H, m), 1.33 (3H, t, J = 7.0 Hz), 1.12 (3H, t, J = 7.2
Hz). 13C NMR (100 MHz, CDCl3): δ 171.66, 169.00, 161.40, 159.65
(d, JC−F = 247.1 Hz), 149.51, 131.95 (d, JC−F = 1.5 Hz), 130.50 (d,
JC−F = 3.0 Hz), 129.03 (d, JC−F = 3.0 Hz), 128.89 (d, JC−F = 8.9 Hz),
128.35 (d, JC−F = 3.0 Hz), 125.67, 124.34 (d, JC−F = 3.8 Hz), 116.20,
115.98, 115.84, 76.77, 60.20, 59.53, 43.66, 31.29, 14.57, 14.01. MS
(ESI, positive) m/z calcd for C22H23FNO5 (M + H): 400.2; found
400.2. HPLC purity: 98.9%.
Methyl 2-Amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-ox-
oethyl)-4H-chromene-3-carboxylate (7b). To a 25 mL flask was
added ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoeth-
yl)-4H-chromene-3-carboxylate (15.5 mg, 0.035 mmol) followed by
methanol (1 mL). Sodium borohydride (6.61 mg, 0.0175 mmol) was
slowly added, and the mixture was stirred at room temperature for 5 h.
The resulting mixture was diluted with 25 mL of DCM and washed
with saturated NH4Cl solution. The aqueous phase was extracted with
DCM (20 mL × 3), and the combined organic layer was washed with
brine and dried over Na2SO4. Solvent evaporation gave a crude oil
which was further purified by column chromatography to yield the
1
pure product. Yield: 21%. H NMR (400 MHz, CDCl3): δ 7.44 (1H,
d, J = 2.1 Hz), 7.39 (1H, dd, J = 8.3, 2.1 Hz), 7.02 (1H, d, J = 8.3 Hz),
6.66 (2H, d, J = 2.2 Hz), 6.45 (1H, t, J = 2.2 Hz), 6.34 (2H, br s), 4.33
(1H, dd, J = 7.1, 4.8 Hz), 4.02 (2H, q, J = 7.1 Hz), 3.84 (6H, s), 3.77
(3H, s), 2.67 (1H, dd, J = 14.8, 4.8 Hz), 2.61 (1H, dd, J = 14.8, 7.1
Hz), 1.13 (3H, t, J = 7.1 Hz). 13C NMR (100 MHz, CDCl3): δ 171.63,
169.40, 161.55, 161.06, 149.60, 142.50, 137.50, 127.06, 126.46, 125.83,
116.06, 105.19, 99.20, 76.58, 60.21, 55.41, 50.98, 43.70, 31.39, 14.08.
MS (ESI, positive) m/z calcd for C23H26NO7 (M + H): 428.2; found
428.2. HPLC purity: 95%.
Oxiran-2-ylmethyl 2-Amino-6-(3,5-dimethoxyphenyl)-4-(2-
(oxiran-2-ylmethoxy)-2-oxoethyl)-4H-chromene-3-carboxylate
(7j). To a stirred solution of 7f (0.17 mmol) in dry DCM under
nitrogen at 0 °C was added 0.37 mmol of m-CPBA over a period of 5
min. After 1 h, the ice bath was removed and the mixture was left to
reach room temperature over 24 h. The reaction mixture was extracted
with sodium bicarbonate, washed with brine, dried over Na2SO4,
filtered, and concentrated. The residue was purified by chromatog-
raphy to afford the pure compound. Yield: 48%. 1H NMR (400 MHz,
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(3-fluorophenyl)-
4H-chromene-3-carboxylate (8c). Yield: 31%. 1H NMR (400
MHz, CDCl3): δ 7.60−7.20 (5H, m), 7.10−7.00 (2H, m), 6.37 (2H,
br s), 4.42−4.34 (1H, m), 4.32−4.20 (2H, m), 4.10−4.00 (2H, m),
2.77−2.60 (2H, m), 1.36 (3H, t, J = 7.2 Hz), 1.12 (3H, t, J = 7.2 Hz).
13C NMR (100 MHz, CDCl3): δ 171.66, 168.96, 163.14 (d, JC−F
=
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244.8 Hz), 161.35, 149.80, 142.52 (d, JC−F = 8.2 Hz), 136.13, 130.21
(d, JC−F = 8.2 Hz), 127.06, 126.31, 126.08, 122.41 (d, JC−F = 3.0 Hz),
116.25, 113.92 (d, JC−F = 21.6 Hz), 113.68 (d, JC−F = 21.6 Hz), 76.62,
60.22, 59.55, 43.60, 31.29, 14.55, 14.03. MS (ESI, positive) m/z calcd
for C22H23FNO5 (M + H): 400.2; found 400.2. HPLC purity: 98. 9%.
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(4-fluorophenyl)-
4H-chromene-3-carboxylate (8d). Yield: 42%. 1H NMR (400
MHz, CDCl3): δ 7.55−7.44 (2H, m), 7.42 (1H, d, J = 2.1 Hz), 7.35
(1H, dd, J = 8.4, 2.1 Hz), 7.15−7.05 (2H, m), 7.01 (1H, d, J = 8.4 Hz),
6.34 (2H, br s), 4.35 (1H, dd, J = 6.7, 4.1 Hz), 4.24 (2H, q, J = 7.0
Hz), 4.02 (2H, q, J = 7.0 Hz), 2.75−2.55 (2H, m), 1.33 (3H, t, J = 7.0
Hz), 1.12 (3H, t, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3): δ 171.69,
168.96, 162.33 (d, JC−F = 244.1 Hz), 161.40, 149.39, 136.46, 136.41 (d,
JC−F = 2.2 Hz), 128.35 (d, JC−F = 9.0 Hz), 126.92, 126.20, 125.99,
116.15, 115.59 (d, JC−F = 21.6 Hz), 76.65, 60.19, 59.53, 43.64, 31.30,
14.55, 14.03. MS (ESI, positive) m/z calcd for C22H23FNO5 (M+1):
400.2; found 400.2. HPLC purity: 98.5%.
124.56, 122.81, 116.27, 76.60, 60.27, 59.55, 43.60, 31.25, 14.57, 14.08.
MS (ESI, positive) m/z calcd for C20H22NO5S (M + H): 388.1; found
388.1. HPLC purity: 95.6%.
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(pyridin-2-yl)-4H-
chromene-3-carboxylate (8i). Yield: 62%. 1H NMR (400 MHz,
CDCl3): δ 8.69−8.61 (1H, m), 7.90 (1H, d, J = 2.0 Hz), 7.85 (1H, dd,
J = 8.4, 2.0 Hz), 7.75−7.63 (2H, m), 7.22−7.16 (1H, m), 7.04 (1H, d,
J = 8.4 Hz), 6.36 (2H, br s), 4.37 (1H, dd, J = 6.6, 4.8 Hz), 4.23 (2H,
q, J = 7.1 Hz), 4.01 (2H, q, J = 7.1 Hz), 2.70 (1H, dd, J = 15.0, 4.8
Hz), 2.65 (1H, dd, J = 15.0, 6.6 Hz), 1.32 (3H, t, J = 7.1 Hz), 1.12
(3H, t, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3): δ 171.70, 169.03,
161.40, 156.49, 150.71, 149.61, 136.74, 135.65, 127.04, 126.29, 125.91,
121.93, 120.06, 116.15, 76.63, 60.22, 59.53, 43.53, 31.34, 14.58, 14.04.
MS (ESI, positive) m/z calcd for C21H23N2O5 (M + H): 383.2; found
383.2. HPLC purity: 99.7%.
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(pyridin-3-yl)-4H-
chromene-3-carboxylate (8j). Yield: 69%. 1H NMR (400 MHz,
CDCl3): δ 8.79 (1H, d, J = 1.6 Hz), 8.57 (1H, dd, J = 5.0, 1.6 Hz),
7.81 (1H, dt, J = 7.9, 1.6 Hz), 7.47 (1H, d, J = 2.2 Hz), 7.40 (1H, dd, J
= 8.4, 2.2 Hz), 7.34 (1H, dd, J = 7.9, 5.0 Hz), 7.06 (1H, d, J = 8.4 Hz),
6.37 (2H, br s), 4.36 (1H, dd, J = 7.2, 4.3 Hz), 4.29−4.19 (2H, m),
4.02 (2H, q, J = 7.2 Hz), 2.69 (1H, dd, J = 15.0, 4.3 Hz), 2.62 (1H, dd,
J = 15.0, 7.2 Hz), 1.33 (3H, t, J = 7.1 Hz), 1.12 (3H, t, J = 7.2 Hz). 13C
NMR (100 MHz, CDCl3): δ 171.63, 168.90, 161.31, 150.03, 148.35,
148.00, 135.76, 134.02, 133.99, 127.16, 126.42, 126.39, 123.50, 116.52,
76.57, 60.23, 59.56, 43.58, 31.24, 14.55, 14.05. MS (ESI, positive) m/z
calcd for C21H23N2O5 (M + H): 383.2; found 383.2. HPLC purity:
99.8%.
Ethyl 2-Amino-6-(2,4-difluorophenyl)-4-(2-ethoxy-2-oxoeth-
1
yl)-4H-chromene-3-carboxylate (8e). Yield: 53%. H NMR (400
MHz, CDCl3): δ 7.42−7.28 (3H, m), 7.02 (1H, d, J = 8.4 Hz), 6.97−
6.81 (2H, m), 6.35 (2H, br s), 4.34 (1H, dd, J = 7.4, 4.3 Hz), 4.23 (2H,
q, J = 7.0 Hz), 4.02 (2H, q, J = 7.2 Hz), 2.75−2.55 (2H, m), 1.32 (3H,
t, J = 7.0 Hz), 1.13 (3H, t, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3):
δ 171.64, 168.94, 162.15 (dd, JC−F = 247.0, 11.9 Hz), 159.60 (dd, JC−F
= 248.9, 11.5 Hz), 161.37, 149.53, 131.15 (t, JC−F = 8.2 Hz), 131.14 (d,
JC−F = 9.6 Hz), 128.92 (d, JC−F = 2.2 Hz), 128.20 (d, JC−F = 3.0 Hz),
125.79, 124.45 (dd, JC−F = 13.4, 3.8 Hz), 115.93, 111.53 (dd, JC−F
=
20.8, 3.8 Hz), 104.34 (t, JC−F = 25.7 Hz), 76.69, 60.19, 59.54, 43.63,
31.24, 14.54, 14.00. MS (ESI, positive) m/z calcd for C22H22F2NO5
(M + H): 418.2; found 418.2. HPLC purity: 96.1%.
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(pyridin-4-yl)-4H-
1
chromene-3-carboxylate (8k). Yield: 41%. H NMR (400 MHz,
CDCl3): δ 8.63 (2H, d, J = 4.7 Hz), 7.55 (1H, d, J = 2.0 Hz), 7.50−
7.40 (3H, m), 7.06 (1H, d, J = 8.4 Hz), 6.38 (2H, br s), 4.36 (1H, dd, J
= 7.3, 4.4 Hz), 4.23 (2H, q, J = 7.0 Hz), 4.02 (2H, q, J = 7.0 Hz), 2.69
(1H, dd, J = 15.2, 4.4 Hz), 2.61 (1H, dd, J = 15.2, 7.3 Hz), 1.33 (3H, t,
J = 7.0 Hz), 1.12 (3H, t, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3): δ
171.63, 168.85, 161.20, 150.70, 150.22, 147.33, 134.16, 127.16, 126.47,
126.29, 121.23, 116.59, 76.55, 60.25, 59.61, 43.55, 31.20, 14.54, 14.04.
MS (ESI, positive) m/z calcd for C21H23N2O5 (M + H): 383.2; found
383.2. HPLC purity: 99.9%.
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(pyrimidin-3,5-yl)-
4H-chromene-3-carboxylate (8l). Yield: 38%. 1H NMR (400 MHz,
CDCl3): δ 9.19 (1H, s), 8.91 (2H, s), 7.50 (1H, d, J = 2.2 Hz), 7.41
(1H, dd, J = 8.4, 2.2 Hz), 7.11 (1H, d, J = 8.4 Hz), 6.36 (2H, br s),
4.37 (1H, dd, J = 7.3, 4.2 Hz), 4.30−4.18 (2H, m), 4.03 (2H, q, J = 7.1
Hz), 2.70 (1H, dd, J = 15.0, 4.2 Hz), 2.63 (1H, dd, J = 15.0, 7.3 Hz),
1.33 (3H, t, J = 7.1 Hz), 1.14 (3H, t, J = 7.1 Hz). 13C NMR (100 MHz,
CDCl3): δ 171.57, 168.81, 161.13, 157.36, 154.61, 150.72, 133.54,
130.36, 127.11, 127.00, 126.32, 116.98, 76.53, 60.31, 59.66, 43.49,
31.12, 14.56, 14.10. MS (ESI, positive) m/z calcd for C20H22N3O5 (M
+ H): 384.2; found 384.2. HPLC purity: 98.4%.
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(2-methoxypyri-
din-4-yl)-4H-chromene-3-carboxylate (8m). Yield: 44%. 1H
NMR (400 MHz, CDCl3): δ 8.18 (1H, d, J = 5.5 Hz), 7.51 (1H, d,
J = 2.2 Hz), 7.43 (1H, dd, J = 8.4, 2.2 Hz), 7.07−7.00 (2H, m), 6.88
(1H, d, J = 0.8 Hz), 6.36 (2H, br s), 4.34 (1H, dd, J = 7.2, 4.3 Hz),
4.28−4.18 (2H, m), 4.01 (2H, q, J = 7.1 Hz), 3.97 (3H, s), 2.68 (1H,
dd, J = 15.2, 4.3 Hz), 2.61 (1H, dd, J = 15.2, 7.2 Hz), 1.32 (3H, t, J =
7.1 Hz), 1.12 (3H, t, J = 7.1 Hz). 13C NMR (100 MHz, CDCl3): δ
171.58, 168.88, 164.89, 161.23, 150.59, 150.15, 147.22, 134.34, 127.10,
126.28, 126.27, 116.41, 114.98, 108.02, 76.55, 60.23, 59.57, 53.49,
43.53, 31.21, 14.55, 14.04. MS (ESI, positive) m/z calcd for
C22H25N2O6 (M + H): 413.2; found 413.2. HPLC purity: 99.8%.
Ethyl 2-Amino-6-(3-aminophenyl)-4-(2-ethoxy-2-oxoethyl)-
4H-chromene-3-carboxylate (9a). Yield: 63%. 1H NMR (400
MHz, CDCl3) δ 7.45 (1H, d, J = 2.26 Hz), 7.39 (1H, dd, J = 2.26, 8.53
Hz), 7.21 (1H, t, J = 7.78 Hz), 7.00 (1H, d, J = 8.53 Hz), 6.94 (1H, d, J
= 7.78 Hz), 6.87 (1H, t, J = 1.88 Hz), 6.68 (1H, dd, J = 2.01, 7.78 Hz),
6.33 (2H, br s), 4.35 (1H, dd, J = 4.52, 7.03 Hz), 4.24 (2H, q, J = 7.11
Hz), 4.02 (2H, q, J = 7.19 Hz), 2.69 (1H, dd, J = 4.52, 15.00 Hz), 2.63
(1H, dd, J = 7.03, 15.00 Hz), 1.34 (3H, t, J = 7.15 Hz), 1.13 (3H, t, J =
Ethyl 2-Amino-6-(3,5-difluorophenyl)-4-(2-ethoxy-2-oxoeth-
1
yl)-4H-chromene-3-carboxylate (8f). Yield: 69%. H NMR (400
MHz, CDCl3): δ 7.44 (1H, d, J = 2.2 Hz), 7.36 (1H, dd, J = 8.4, 2.2
Hz), 7.08−6.99 (3H, m), 6.76 (1H, tt, J = 8.9 Hz, 2.3 Hz), 6.34 (2H,
br s), 4.35 (1H, dd, J = 7.2, 4.3 Hz), 4.24 (2H, q, J = 7.1 Hz), 4.03
(2H, q, J = 7.1 Hz), 2.69 (1H, dd, J = 15.0, 4.3 Hz), 2.63 (1H, dd, J =
15.0, 7.2 Hz), 1.33 (3H, t, J = 7.1 Hz), 1.13 (3H, t, J = 7.1 Hz). 13C
NMR (100 MHz, CDCl3): δ 171.62, 168.89, 163.28 (dd, JC−F = 247.0,
13.4 Hz), 161.25, 150.21, 143.57 (t, JC−F = 9.7 Hz), 135.03 (t, JC−F
=
2.6 Hz), 127.07, 126.29, 126.25, 116.40, 109.60 (dd, JC−F = 18.6, 7.4
Hz), 102.36 (t, JC−F = 25.3 Hz), 76.57, 60.25, 59.59, 43.54, 31.23,
14.54, 14.04. MS (ESI, positive) m/z calcd for C22H22F2NO5 (M +
H): 418.2; found 418.2. HPLC purity: 96.3%.
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(furan-2-yl)-4H-
chromene-3-carboxylate (8g). This compound was synthesized
by Suzuki−Miyaura coupling of 8a with 2-furylboronic acid MIDA
ester. (Refer to the general procedure for the synthesis of 6-
arylcoumarins.) Yield: 15%. 1H NMR (400 MHz, CDCl3): δ 7.55 (1H,
d, J = 2.1 Hz), 7.49 (1H, dd, J = 8.6, 2.1 Hz), 7.43 (1H, dd, J = 2.0, 0.7
Hz), 6.97 (1H, d, J = 8.6 Hz), 6.56 (1H, dd, J = 3.3, 0.7 Hz), 6.45 (1H,
dd, J = 3.3, 2.0 Hz), 6.31 (2H, br s), 4.31 (1H, dd, J = 7.1, 4.4 Hz),
4.23 (2H, q, J = 7.0 Hz), 4.03 (2H, q, J = 7.2 Hz), 2.68 (1H, dd, J =
14.8, 4.4 Hz), 2.61 (1H, dd, J = 14.8, 7.1 Hz), 1.33 (3H, t, J = 7.0 Hz),
1.14 (3H, t, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3): δ 171.64,
168.99, 161.32, 153.31, 149.18, 141.87, 127.40, 125.91, 123.75, 123.28,
116.12, 111.62, 104.46, 76.60, 60.23, 59.53, 43.54, 31.29, 14.55, 14.03.
MS (ESI, positive) m/z calcd for C20H22NO6 (M + H): 372.1; found
372.1. HPLC purity: 98.1%.
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(thiophen-2-yl)-
4H-chromene-3-carboxylate (8h). This compound was synthesized
by Suzuki−Miyaura coupling of 8a with 2-thiopheneboronic acid
MIDA ester. (Refer to the general procedure for the synthesis of 6-
arylcoumarins.) Yield: 25%. 1H NMR (400 MHz, CDCl3): δ 7.48 (1H,
d, J = 2.2 Hz), 7.43 (1H, dd, J = 8.4, 2.2 Hz), 7.25 (1H, dd, J = 5.1, 1.0
Hz), 7.23 (1H, dd, J = 3.6, 1.0 Hz), 7.06 (1H, dd, J = 5.1, 3.6 Hz), 6.97
(1H, d, J = 8.4 Hz), 6.32 (2H, br s), 4.32 (1H, dd, J = 7.2, 4.3 Hz),
4.23 (2H, q, J = 7.1 Hz), 4.03 (2H, q, J = 7.2 Hz), 2.68 (1H, dd, J =
14.8, 4.3 Hz), 2.61 (1H, dd, J = 14.8, 7.2 Hz), 1.33 (3H, t, J = 7.1 Hz),
1.15 (3H, t, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3): δ 171.63,
168.97, 161.32, 149.36, 143.56, 130.83, 128.00, 126.11, 125.81, 125.30,
5577
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7.15 Hz). 13C NMR (100 MHz, CDCl3) δ 171.7, 169.1, 161.5, 149.4,
146.7, 141.4, 137.6, 129.7, 126.9, 126.3, 125.7, 117.3, 116.0, 114.0,
113.5, 60.2, 59.5, 43.6, 31.3, 14.6, 14.0 (two coinciding carbon
resonances). MS (ESI, positive) m/z calcd for C22H24N2O5 (M + H):
397.17; found, 397.07. HPLC purity: 95.5%.
Ethyl 2-Amino-6-(3-aminophenyl)-4-(2-oxo-2-(prop-2-yn-1-
yloxy)ethyl)-4H-chromene-3-carboxylate (9b). Yield: 29%. 1H
NMR (400 MHz, CDCl3): δ 7.45 (1H, d, J = 2 Hz), 7.38 (1H, dd, J =
2.4, 8.4 Hz), 7.19 (1H, t, J = 8 Hz), 7.00 (1H, d, J = 8.4 Hz), 6.93−
6.91 (1H, m), 6.85 (1H, t, J = 2 Hz), 6.66−6.64 (1H, m), 6.34 (2H, s),
4.58 (2H, t, J = 2.4 Hz), 4.39−4.37 (1H, m), 4.24 (2H, q, J = 6.8 Hz),
3.74 (2H, s), 2.76−2.62 (2H, m), 2.28 (1H, t, J = 2.4 Hz), 1.33 (3H, t,
J = 7.2 Hz). 13C NMR (100 MHz, CDCl3): δ 170.99, 169.07, 161.68,
149.45, 146.85, 141.58, 137.92, 129.78, 127.09, 126.59, 125.58, 117.56,
116.21, 114.09, 113.82, 77.74, 76.76, 74.99, 59.71, 51.86, 43.53, 31.45,
14.72. MS (ESI, positive) m/z calcd for C23H22N2O5 (M + H):
407.15; found 407.05. HPLC purity: 99.72%.
Prop-2-yn-1-yl 2-Amino-6-(3-aminophenyl)-4-(2-oxo-2-
(prop-2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxylate (9c).
Yield: 59%. 1H NMR (400 MHz, CDCl3): δ 7.45 (1H, d, J = 2
Hz), 7.38 (1H, dd, J = 2, 8.4 Hz), 7.19 (1H, t, J = 8 Hz), 7.01 (1H, d, J
= 8.4 Hz), 6.92 (1H, d, J = 7.6 Hz), 6.85 (1H, t, J = 2 Hz), 6.66 (1H,
dd, J = 1.6, 8 Hz), 6.41 (2H, s), 4.79 (2H, t, J = 2.2 Hz), 4.60−4.59
(2H, m), 4.41−4.38 (1H, m), 3.74 (2H, s), 2.78−2.64 (2H, m), 2.47
(1H, t, J = 2.0 Hz), 2.29 (1H, t, J = 2.6 Hz). 13C NMR (100 MHz,
CDCl3): δ 170.81, 167.97, 162.35, 149.26, 146.85, 141.41, 138.02,
129.75, 127.02, 126.63, 125.33, 117.45, 116.18, 114.11, 113.75, 78.83,
77.71, 75.89, 75.04, 74.41, 51.89, 51.21, 43.47, 31.23. MS (ESI,
positive) m/z calcd for C24H20N2O5 (M + H): 417.14; found 417.1.
HPLC purity: 99.5%.
2.4 Hz), 4.39−4.37 (1H, m), 3.72 (2H, s), 2.77−2.65 (2H, m), 2.47
(1H, t, J = 2 Hz), 2.28 (1H, t, J = 2.2 Hz). 13C NMR (100 MHz,
CDCl3): δ 170.88, 168.07, 162.47, 148.68, 145.95, 137.97, 130.71,
127.98, 126.27, 125.97, 125.39, 116.23, 115.47, 78.87, 77.77, 76.06,
74.97, 74.37, 51.92, 51.25, 43.53, 31.32. MS (ESI, positive) m/z calcd
for C24H20N2O5 (M + H): 417.14; found 417.2. HPLC purity: 96.9%.
Ethyl 2-Amino-4-(2-ethoxy-2-oxoethyl)-6-(3-(methylamino)-
1
phenyl)-4H-chromene-3-carboxylate (9h). Yield: 74%. H NMR
(400 MHz, CDCl3): δ 7.46 (1H, d, J = 2 Hz), 7.39 (1H, dd, J = 2.1, 8.4
Hz), 7.22 (1H, t, J = 8 Hz), 7.00 (1H, d, J = 8.4 Hz), 6.87 (1H, d, J =
7.6 Hz), 6.75 (1H, s), 6.59 (1H, dd, J = 2.1, 8 Hz), 6.38 (2H, s), 4.36
(1H, m), 4.24 (2H, q, J = 7.0 Hz), 4.03 (2H, q, J = 7.2 Hz), 3.74 (1H,
br s), 2.88 (3H, s), 2.72−2.60 (2H, m), 1.34 (3H, t, J = 7.2 Hz), 1.13
(3H, t, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3): δ 171.83, 169.16,
161.65, 149.78, 149.41, 141.47, 138.14, 129.65, 127.07, 126.47, 125.78,
116.14, 116.01, 111.49, 110.88, 76.74, 60.26, 59.59, 43.79, 31.47,
30.85, 14.67, 14.15. MS (ESI, positive) m/z calcd for C23H26N2O5 (M
+ H): 411.18; found 411.2. HPLC purity: 99.2%.
Ethyl 2-Amino-6-(3-(methylamino)phenyl)-4-(2-oxo-2-
(prop-2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxylate (9i).
Yield: 23%. 1H NMR (400 MHz, CDCl3): δ 7.46 (1H, d, J = 2
Hz), 7.41 (1H, dd, J = 2, 8.4 Hz), 7.22 (1H, t, J = 8 Hz), 7.00 (1H, d, J
= 8.2 Hz), 6.86 (1H, d, J = 7.6 Hz), 6.75 (1H, s), 6.58 (1H, dd, J = 1.8,
8 Hz), 6.34 (2H, s), 4.58 (2H, t, J = 2.5 Hz), 4.40−4.37 (1H, m), 4.24
(2H, q, J = 7.0 Hz), 3.81 (1H, s), 2.89 (3H, s), 2.76−2.62 (2H, m),
2.27 (1H, t, J = 2.4 Hz), 1.33 (3H, t, J = 7.0 Hz). 13C NMR (100 MHz,
CDCl3): δ 170.99, 169.09, 161.71, 149.78, 149.41, 141.53, 138.36,
129.66, 127.16, 126.70, 125.54, 116.37, 116.17, 111.50, 111.51, 77.73,
76.77, 74.98, 59.71, 51.86, 43.53, 31.47, 30.93, 14.73. MS (ESI,
positive) m/z calcd for C24H24N2O5 (M + H): 421.17; found 421.2.
HPLC purity: 97.2%.
Prop-2-yn-1-yl 2-(2-Amino-6-(3-aminophenyl)-3-cyano-4H-
chromen-4-yl)acetate (9d). This compound was synthesized by
following the same general procedure except that malononitrile was
Prop-2-yn-1-yl 2-Amino-6-(3-(methylamino)phenyl)-4-(2-
oxo-2-(prop-2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxylate
1
(9j). Yield: 63%. H NMR (400 MHz, CDCl3): δ 7.47 (1H, d, J = 2
1
used instead of ethyl cyanoacetate. Yield: 61%. H NMR (400 MHz,
Hz), 7.40 (1H, dd, J = 2.2, 8.6 Hz), 7.23 (1H, t, J = 8 Hz), 7.01 (1H, d,
J = 8.4 Hz), 6.86 (1H, d, J = 7.6 Hz), 6.74 (1H, s), 6.59 (1H, dd, J = 2,
8.1 Hz), 6.40 (2H, s), 4.79 (2H, t, J = 2 Hz), 4.59 (2H, t, J = 2.8 Hz),
4.39 (1H, m), 3.80 (1H, br s), 2.89 (3H, s), 2.78−2.66 (2H, m), 2.47
(1H, t, J = 2.4 Hz), 2.28 (1H, t, J = 2.8 Hz). 13C NMR (100 MHz,
CDCl3): δ 170.82, 168.03, 162.40, 149.77, 149.28, 141.41, 138.52,
129.68, 127.14, 126.77, 125.33, 116.32, 116.18, 111.54, 111.09, 78.85,
77.73, 75.99, 75.01, 74.39, 51.91, 51.25, 43.49, 31.29, 30.91. MS (ESI,
positive) m/z calcd for C25H22N2O5 (M + H): 431.15; found 431.3.
HPLC purity: 97.5%.
CDCl3): δ 7.41 (1H, dd, J = 2, 8.4 Hz), 7.39 (1H, d, J = 2 Hz), 7.20
(1H, t, J = 7.8 Hz), 7.01 (1H, d, J = 8.4 Hz), 6.92 (1H, d, J = 8 Hz),
6.83 (1H, s), 6.66 (1H, dd, J = 1.8, J = 7.8 Hz), 4.67 (2H, d, J = 2 Hz),
4.66 (2H, s), 4.16−4.11 (1H, m), 3.76 (2H, s), 2.81−2.77 (2H, m),
2.35 (1H, t, J = 2.4 Hz). 13C NMR (100 MHz, CDCl3): δ 170.07,
160.97, 148.71, 146.95, 141.16, 138.76, 129.93, 127.34, 126.72, 122.42,
119.69, 117.54, 116.81, 114.40, 113.76, 77.36, 75.29, 58.05, 52.35,
43.25, 32.17. MS (ESI, positive) m/z calcd for C21H17N3O3 (M + H):
360.13; found 360.1. HPLC purity: 95%.
Ethyl 2-Amino-6-(4-aminophenyl)-4-(2-ethoxy-2-oxoethyl)-
4H-chromene-3-carboxylate (9e). Yield: 72%. 1H NMR (400
MHz, CDCl3): δ 7.39 (1H, d, J = 2 Hz), 7.35−7.32 (3H, m), 6.97
(1H, d, J = 8.4 Hz), 6.72 (2H, d, J = 8.4 Hz), 6.33 (2H, s), 4.35−4.32
(1H, m), 4.23 (2H, q, J = 7.2 Hz), 4.01 (2H, q, J = 7.2 Hz), 3.73 (2H,
s), 2.70−2.58 (2H, m), 1.33 (3H, t, J = 7.2 Hz), 1.12 (3H, t, J = 7.2
Hz). 13C NMR (100 MHz, CDCl3): δ 171.91, 169.21, 161.69, 148.81,
145.89, 137.61, 130.75, 127.81, 126.23, 125.83, 125.67, 116.08, 115.49,
60.29, 59.61, 43.85, 31.53, 14.70, 14.18 (two coinciding carbon
resonances). MS (ESI, positive) m/z calcd for C22H24N2O5 (M + H):
397.17; found 397.2. HPLC purity: 99.4%.
Ethyl 2-Amino-6-(4-aminophenyl)-4-(2-oxo-2-(prop-2-yn-1-
yloxy)ethyl)-4H-chromene-3-carboxylate (9f). Yield: 22%. 1H
NMR (400 MHz, CDCl3): δ 7.40 (1H, d, J = 2 Hz), 7.35 (3H, d, J =
8.4 Hz), 6.99 (1H, d, J = 8.4 Hz), 6.73 (2H, d, J = 8.4 Hz), 6.31 (2H,
s), 4.58 (2H, t, J = 2.8 Hz), 4.38−4.35 (1H, m), 4.24 (2H, q, J = 7.2
Hz), 3.72 (2H, s), 2.76−2.62 (2H, m), 2.27 (1H, t, J = 2.4 Hz), 1.33
(3H, t, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3): δ 171.04, 169.14,
161.78, 148.82, 145.90, 137.80, 130.85, 128.00, 126.30, 125.91, 125.59,
116.22, 115.47, 77.77, 77.37, 74.93, 59.70, 51.86, 43.56, 31.50, 14.75.
MS (ESI, positive) m/z calcd for C23H22N2O5 (M + H): 407.15; found
407.05. HPLC purity: 94.4%.
Prop-2-yn-1-yl 2-Amino-6-(3-(dimethylamino)phenyl)-4-(2-
oxo-2-(prop-2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxylate
(9k). Yield: 60%. 1H NMR (400 MHz, CDCl3): δ 7.48 (1H, d, J = 2.1
Hz), 7.43 (1H, dd, J = 2.2, 8.5 Hz), 7.28 (1H, t, J = 8 Hz), 7.02 (1H, d,
J = 8.4 Hz), 6.89−6.85 (2H, m), 6.72 (1H, dd, J = 2, 8.0 Hz), 6.40
(2H, s), 4.79 (2H, t, J = 2 Hz), 4.59 (2H, t, J = 2.8 Hz), 4.42−4.39
(1H, m), 3.0 (6H, s), 2.78−2.67 (2H, m), 2.47 (1H, t, J = 2.4 Hz),
2.27 (1H, t, J = 2.8 Hz). 13C NMR (100 MHz, CDCl3): δ 170.84,
168.08, 162.45, 151.08, 149.31, 141.37, 139.00, 129.55, 127.31, 126.95,
125.37, 116.19, 115.77, 111.74, 111.46, 78.86, 77.73, 76.07, 74.99,
74.39, 51.94, 51.29, 43.50, 40.85, 30.32. MS (ESI, positive) m/z calcd
for C26H24N2O5 (M + H): 445.17; found 445.2. HPLC purity: 95.2%.
Ethyl 2-Amino-6-(3-(ethylamino)phenyl)-4-(2-oxo-2-(prop-
2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxylate (9l). Yield:
1
21%. H NMR (400 MHz, CDCl3): δ 7.45 (1H, d, J = 2 Hz), 7.40
(1H, dd, J = 2, 8.4 Hz), 7.21 (1H, t, J = 7.8 Hz), 7.00 (1H, d, J = 8.2
Hz), 6.85 (1H, d, J = 7.7 Hz), 6.75 (1H, s), 6.58 (1H, dd, J = 2.2, 8
Hz), 6.32 (2H, s), 4.57 (2H, t, J = 2.8 Hz), 4.37 (1H, m), 4.23 (2H, q,
J = 7.5 Hz), 3.66 (1H, s), 3.21 (2H, q, J = 7.0 Hz), 2.77−2.57 (2H, m),
2.26 (1H, t, J = 2.3 Hz), 1.32 (3H, t, J = 7.2 Hz), 1.27 (3H, t, J = 7.2
Hz). 13C NMR (100 MHz, CDCl3): δ 171.01, 169.12, 161.72, 149.42,
148.91, 141.55, 138.39, 129.71, 127.16, 126.70, 125.55, 116.35, 116.18,
111.81, 111.52, 77.74, 77.36, 74.98, 59.73, 51.86, 43.54, 38.68, 31.48,
15.07, 14.75. MS (ESI, positive) m/z calcd for C25H26N2O5 (M + H):
435.18; found 435.2. HPLC purity: 98.6%.
Prop-2-yn-1-yl 2-Amino-6-(4-aminophenyl)-4-(2-oxo-2-
(prop-2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxylate (9g).
1
Yield: 67%. H NMR (400 MHz, CDCl3): δ 7.41 (1H, d, J = 1.8
Hz), 7.35 (3H, d, J = 8.4 Hz), 7.00 (1H, d, J = 8.4 Hz), 6.73 (2H, d, J
= 8.4 Hz), 6.39 (2H, s), 4.35−4.79 (2H, t, J = 2 Hz), 4.58 (2H, t, J =
Prop-2-yn-1-yl 2-Amino-6-(3-ethylamino)phenyl)-4-(2-oxo-
2-(prop-2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxylate (9m).
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1
Yield: 61%. H NMR (400 MHz, CDCl3): δ 7.46 (1H, d, J = 2 Hz),
s), 7.71−7.64 (3H, m), 7.56 (1H, J = 2.0 Hz), 7.47 (1H, dd, J = 2, 8.4
Hz), 7.10 (1H, d, J = 8.4 Hz), 6.4 (2H, br s), 4.81−4.80 (2H, m),
4.66−4.56 (2H, m), 4.43−4.41 (1H, m), 2.81−2.68 (2H, m), 2.47
(1H, t, J = 2.0 Hz), 2.29 (1H, t, J = 2.4 Hz). 13C NMR (100 MHz,
CDCl3): δ 170.84, 167.93, 162.17, 150.29, 142.91, 140.68, 135.72,
130.77, 128.66, 127.54, 126.92, 126.17, 119.92, 119.88, 116.89, 78.73,
77.36, 75.95, 75.00, 74.51, 52.02, 51.41, 43.45, 31.19 (two coinciding
carbon resonances). MS (ESI, positive) m/z calcd for C25H19N5O5 (M
+ H): 470.14; found 470. HPLC purity: 99.1%.
Prop-2-yn-1-yl 2-Amino-6-(4-(methylamino)phenyl)-4-(2-
oxo-2-(prop-2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxylate
(9r). Yield: 35%. 1H NMR (400 MHz, CDCl3): δ 7.41−7.34 (4H, m),
6.98 (1H, d, J = 8.4 Hz), 6.66 (2H, d, J = 8.4 Hz), 6.39 (2H, br s), 4.78
(2H, t, J = 2.4 Hz), 4.59 (2H, t, J = 2.8 Hz), 4.39−4.34 (1H, m), 3.8
(1H, br s), 2.87 (3H, s), 2.77−2.65 (2H, m), 2.46 (1H, t, J = 2.4 Hz),
2.28 (1H, t, J = 2.4 Hz). 13C NMR (100 MHz, CDCl3): δ 170.89,
168.09, 162.50, 148.85, 148.52, 138.13, 129.28, 127.87, 126.11, 125.84,
125.36, 116.20, 112.74, 78.89, 77.78, 76.07, 74.97, 74.36, 51.91, 51.23,
43.54, 31.34, 30.87. MS (ESI, positive) m/z calcd for C25H22N2O5 (M
+ H): 431.15; found 431. HPLC purity: 95.5%.
7.39 (1H, dd, J = 2.2, 8.6 Hz), 7.21 (1H, t, J = 8 Hz), 7.00 (1H, d, J =
8.4 Hz), 6.86 (1H, d, J = 7.6 Hz), 6.74 (1H, s), 6.58 (1H, dd, J = 2, 8
Hz), 6.41 (2H, s), 4.79 (2H, t, J = 2 Hz), 4.59 (2H, t, J = 2.6 Hz), 4.39
(1H, m), 3.65 (1H, br s), 3.21 (2H, q, J = 7.4 Hz), 2.77−2.66 (2H, m),
2.48 (1H, t, J = 2.2 Hz), 2.28 (1H, t, J = 2.4 Hz), 1.28 (3H, t, J = 7.2
Hz). 13C NMR (100 MHz, CDCl3): δ 170.81, 168.01, 162.39, 149.23,
148.85, 141.37, 138.48, 129.67, 127.08, 126.71, 125.29, 116.25, 116.14,
111.83, 111.44, 78.83, 77.71, 75.93, 75.01, 74.39, 51.89, 51.23, 43.47,
38.60, 31.26, 14.98. MS (ESI, positive) m/z calcd for C26H24N2O5 (M
+ H): 445.17; found 445.2. HPLC purity: 99%.
Prop-2-yn-1-yl 2-Amino-6-(3-((2-hydroxyethyl)amino)-
phenyl)-4-(2-oxo-2-(prop-2-yn-1-yloxy)ethyl)-4H-chromene-3-
carboxylate (9n). To the amine 9c (1 mmol) in dry acetonitrile, 2-
iodoethanol (1 mmol) was added. The mixture was refluxed at 80 °C
overnight. It was concentrated followed by the usual workup, giving
the crude mass which upon column purification furnished the
1
monosubstituted product without any disubstitution. Yield: 58%. H
NMR (400 MHz, CDCl3): δ 7.46 (1H, d, J = 2 Hz), 7.40 (1H, dd, J =
2.4, 8.4 Hz), 7.23 (2H, t, J = 8.0 Hz), 7.02 (1H, d, J = 8.4 Hz), 6.90
(1H, d, J = 8 Hz), 6.35 (1H, dd, J = 2.0, 8.0 Hz), 6.41 (2H, br s), 4.79
(2H, t, J = 2 Hz), 4.59 (2H, t, J = 2.4 Hz), 4.41−4.38 (1H, m), 4.13
(1H, br s), 3.88 (2H, t, J = 5.2 Hz), 3.38 (2H, t, J = 5.2 Hz), 2.78−2.66
(2H, m), 2.47 (1H, t, J = 2.4 Hz), 2.29 (1H, t, J = 2.4 Hz). 13C NMR
(100 MHz, CDCl3): δ 170.88, 168.07, 162.41, 149.38, 148.63, 141.59,
138.40, 129.84, 127.22, 126.79, 125.43, 116.99, 116.26, 112.35, 111.99,
78.86, 77.77, 76.09, 75.04, 74.44, 61.48, 51.97, 51.30, 46.29, 43.53,
31.30. MS (ESI, positive) m/z calcd for C26H24N2O6 (M + H):
461.16; found 461.07. HPLC purity: 95.9%.
Prop-2-yn-1-yl 2-Amino-6-(3-(2-chloroacetamido)phenyl)-4-
(2-oxo-2-(prop-2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxy-
late (9o). To an ice cold solution of amine 9c (1 mmol) in dry DCM,
chloroacetyl chloride (1.1 mmol) was added slowly. After the addition,
the ice bath was removed and the contents stirred at room
temperature. Upon completion, the contents were extracted with
DCM, washed with bicarbonate, water, brine, and dried over MgSO4.
Removal of the solvent under reduced pressure gave crude amide
which upon column purification furnished pure product. Yield: 62%.
1H NMR (400 MHz, CDCl3): δ 8.29 (1H, s), 7.71 (1H, s), 7.55−7.47
Prop-2-yn-1-yl 2-Amino-6-(4-(dimethylamino)phenyl)-4-(2-
oxo-2-(prop-2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxylate
(9s). Yield: 37%. 1H NMR (400 MHz, CDCl3): δ 7.45−7.43 (3H, m),
7.37 (1H, dd, J = 2.0, 8.4 Hz), 6.98 (1H, d, J = 8.4 Hz), 6.78 (1H, d, J
= 8.8 Hz), 6.42 (2H, s), 4.79 (2H, t, J = 2.4 Hz), 4.59 2H, t, J = 2.8
Hz), 4.40−4.38 (2H, m), 2.99 (6H, s), 2.78−2.66 (2H, m), 2.47 (1H,
t, J = 2.4 Hz), 2.29 (1H, t, J = 2.4 Hz). 13C NMR (100 MHz, CDCl3):
δ 170.87, 168.06, 162.49, 150.01, 148.48, 137.98, 128.36, 127.63,
126.06, 125.79, 125.34, 116.19, 112.85, 78.89, 77.76, 75.99, 74.97,
74.36, 51.89, 51.19, 43.52, 40.68, 31.31. MS (ESI, positive) m/z calcd
for C26H24N2O5 (M + H): 445.17; found 445.13. HPLC purity: 99.1%.
Ethyl 2-Amino-6-(4-(ethylamino)phenyl)-4-(2-oxo-2-(prop-
2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxylate (9t). Yield:
1
9%. H NMR (400 MHz, CDCl3): δ 7.41−7.34 (4H, m), 6.98 (1H,
d, J = 8.4 Hz), 6.65 (2H, d, J = 8.4 Hz), 6.32 (2H, br s), 4.58 (2H, t, J
= 2.8 Hz), 4.38−4.35 (1H, m), 4.23 (2H, q, J = 7.0 Hz), 3.66 (1H, br
s), 3.19 (2H, q, J = 7.2 Hz), 2.76−2.62 (2H, m), 2.27 (1H, t, J = 2.8
Hz), 1.33 (3H, t, J = 7.2 Hz), 1.27 (3H, t, J = 7.2 Hz). 13C NMR (100
MHz, CDCl3): δ 171.06, 169.15, 161.82, 148.65, 147.93, 137.96,
129.37, 127.92, 126.11, 125.76, 125.55, 116.18, 113.06, 77.79, 77.36,
74.93, 59.69, 51.85, 43.57, 38.65, 31.52, 15.03, 14.75. MS (ESI,
positive) m/z calcd for C25H26N2O5 (M + H): 435.18; found 435.20.
HPLC purity: 97.3%.
(2H, m), 7.43−7.33 (3H, m), 7.04 (1H, d, J = 8.4 Hz), 6.41 (2H, s),
4.79 (2H, m), 4.60 (2H, t, J = 2.5 Hz), 4.40 (1H, m), 4.22 (2H, s),
2.78−2.66 (2H, m), 2.47 (1H, t, J = 2.4 Hz), 2.29 (1H, t, J = 2.4 Hz).
13C NMR (100 MHz, CDCl3): δ 170.82, 168.00, 164.00, 162.32,
149.70, 141.45, 137.27, 137.13, 129.69, 127.26, 126.86, 125.67, 123.99,
119.06, 118.82, 116.48, 78.79, 77.77, 75.98, 74.97, 74.44, 51.97, 51.32,
43.45, 43.04, 31.25. MS (ESI, positive) m/z calcd for C26H21ClN2O6
(M + H): 493.11; found 493.3. HPLC purity: 95.3%.
Prop-2-yn-1-yl 2-Amino-6-(4-(ethylamino)phenyl)-4-(2-oxo-
2-(prop-2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxylate (9u).
Yield: 38%. 1H NMR (400 MHz, CDCl3): δ 7.41−7.34 (4H, m),
6.98 (1H, d, J = 8.4 Hz), 6.65 (2H, d, J = 8.8 Hz), 6.38 (2H, br s), 4.78
(2H, t, J = 2.4 Hz), 4.59 (2H, t, J = 2.8 Hz), 4.39−4.37 (1H, m), 3.62
(1H, br s), 3.19 (2H, q, J = 7.2 Hz), 2.77−2.65 (2H, m), 2.46 (1H, t, J
= 2.4 Hz), 2.28 (1H, t, J = 2.4 Hz), 1.28 (3H, t, J = 7.2 Hz). 13C NMR
(100 MHz, CDCl3): δ 170.87, 168.06, 162.48, 148.48, 147.93, 138.09,
129.17, 127.86, 126.05, 125.78, 125.32, 116.17, 113.03, 78.88, 77.76,
76.01, 74.96, 74.36, 51.89, 51.21, 43.52, 38.58, 31.32, 14.98. MS (ESI,
positive) m/z calcd for C26H24N2O5 (M + H): 445.17; found 417.12.
HPLC purity: 98.6%.
Cell Cultures. HL60, HL60/MX2, CCRF-CEM, and CCRF-
CEM/C2 were purchased from ATCC. HL60/MX2 was developed
from HL60 upon exposure to mitoxantrone, a topoisomerase II
inhibitor. CCRF-CEM/C2 was developed from CCRF-CEM upon
exposure to camptothecin, a topoisomerase I inhibitor. K562, K562/
HHT300, K562/DOX, HL60/ADR, and HL60/DNR cell lines were
provided by Dr. Tang.47 K562/HHT300 was developed from K562
upon exposure to homoharringtonine, an antimicrotubule agent.
K562/DOX was developed from K562 upon exposure to doxorubicin,
a topoisomerase II inhibitor. HL60/ADR and HL60/DNR were
developed from HL60 upon exposure to adriamycin (which is
doxorubicin) and daunorubicin topoisomerase II inhibitors, respec-
tively. HL60 and HL60/DOX cell lines were provided by Dr.
Ganapathi.40 HL60/DOX was developed from HL60 upon exposure
to doxorubicin. CCRF-CEM, CCRF-CEM/VM-1-5, and CCRF-
Ethyl 2-Amino-6-(3-carbamoylphenyl)-4-(2-ethoxy-2-ox-
oethyl)-4H-chromene-3-carboxylate (9p). Yield: 45%. H NMR
1
(400 MHz, CDCl3): δ 8.00 (1H, t, J = 1.5 Hz), 7.74 (1H, dt, J = 7.8,
1.5 Hz), 7.68 (1H, dd, J = 7.7, 1.5 Hz), 7.55−7.45 (2H, m), 7.42 (1H,
dd, J = 8.4, 7.2 Hz), 7.03 (1H, d, J = 8.4 Hz), 6.31 (2H, br s), 5.89
(2H, br s), 4.35 (1H, dd, J = 6.8, 4.5 Hz), 4.23 (2H, q, J = 7.1 Hz),
4.02 (2H, q, J = 7.1 Hz), 2.69 (1H, dd, J = 14.8, 4.5 Hz), 2.63 (1H, dd,
J = 14.8, 6.8 Hz), 1.33 (3H, t, J = 7.1 Hz), 1.12 (3H, t, J = 7.1 Hz). 13C
NMR (100 MHz, CDCl3): δ 171.72, 169.21, 168.96, 161.36, 149.81,
140.83, 136.32, 133.91, 130.27, 129.06, 127.13, 126.43, 126.11, 125.99,
125.86, 116.29, 76.55, 60.25, 59.56, 43.57, 31.32, 14.55, 14.05. MS
(ESI, positive) m/z calcd for C23H25N2O6 (M + H): 425.2; found
425.2. HPLC purity: 99.2%.
Prop-2-yn-1-yl 6-(3-(1H-Tetrazol-1-yl)phenyl)-2-amino-4-(2-
oxo-2-(prop-2-yn-1-yloxy)ethyl)-4H-chromene-3-carboxylate
(9q). To the ionic liquid (EAN, 5 mmol) in a screw-capped vial,
triethyl orthoformate (1.2 mmol), TMSN3 (1 mmol), and amine 9c (1
mmol) were added, and the contents were stirred vigorously at room
temperature for about 1 h. Upon completion, the compound was
extracted in DCM, washed a few times with water, brine, and dried
over MgSO4. The crude product obtained by evaporating the solvent
under reduced pressure was purified by column chromatography.
1
Yield: 43%. H NMR (400 MHz, CDCl3): δ 9.06 (1H, s), 7.87 (1H,
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CEM/VLB100 were provided by Dr. Beck.45,46 CCRF-CEM/VM-1-5
was developed from CCRF-CEM upon exposure to teniposide, a
topoisomerase II inhibitor. CCRF-CEM/VLB100 was developed from
CCRF-CEM upon exposure to vinblastine, an antimicrotubule agent.
As HL60/ADR and HL60/DOX are from two different sources, they
do not behave the same. Similarly even the three parental cell lines for
HL60 and the two parental cell lines for CCRF-CEM do not behave
the same. We therefore have evaluated all the parental cell lines and
the MDR cell lines, using the names as in their original reports.
Comparison has been made only between the MDR cell line with its
corresponding parental cell line. All cell lines were grown in RPMI
1640 purchased from ATCC, supplemented with 10% FBS at 37 °C
with 5% CO2 in air atmosphere.
Cytotoxicity Measurement. In vitro cytotoxicity of small
molecules was assayed by determining their ability to inhibit the
growth of the tumor cells following our established procedures.29
Statistical Analysis. In vitro cytotoxicity assay was performed at
least twice with triplicates in each experiment. Data in Tables 1−4 are
presented as the mean SEM. Data in Tables 5 and 6 are presented as
mean values only.
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Gottesman, M. M. Targeting multidrug resistance in cancer. Nat. Rev.
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Bootman, M. D.; Berridge, M. J.; Conway, S. J.; Holmes, A. B.;
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ASSOCIATED CONTENT
■
(11) Li, C.; Wang, X.; Vais, H.; Thompson, C. B.; Foskett, J. K.;
White, C. Apoptosis regulation by Bcl-x(L) modulation of mammalian
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S
* Supporting Information
HMBC spectra of 9b. This material is available free of charge
via the Internet at http://pubs.acs.org.
(12) Kuo, T. H.; Kim, H. R.; Zhu, L.; Yu, Y.; Lin, H. M.; Tsang, W.
Modulation of endoplasmic reticulum calcium pump by Bcl-2.
Oncogene 1998, 17, 1903−1910.
AUTHOR INFORMATION
■
Corresponding Author
(13) Dremina, E. S.; Sharov, V. S.; Schoneich, C. Displacement of
̈
*Phone: 612-626-5675. Fax: 612-624-0139. E-mail: xingx009@
umn.edu.
SERCA from SR lipid caveolae-related domains by Bcl-2: a possible
mechanism for SERCA inactivation. Biochemistry 2006, 45, 175−184.
(14) Grabowski, D. R.; Dubyak, G. R.; Rybicki, L.; Hidaka, H.;
Ganapathi, R. Tumor cell resistance to topoisomerase II poisons: role
for intracellular free calcium in the sensitization by inhibitors or
calcium-calmodulin-dependent enzymes. Biochem. Pharmacol. 1998,
56, 345−349.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(15) Tombal, B.; Weeraratna, A. T.; Denmeade, S. R.; Isaacs, J. T.
Thapsigargin induces a calmodulin/calcineurin-dependent apoptotic
cascade responsible for the death of prostatic cancer cells. Prostate
2000, 43, 303−317.
We thank the Leukemia Research Fund, Masonic Cancer
Center, University of Minnesota (LRF Seed Grant, C.X.); the
Faculty Development Grant, University of Minnesota (FDG
Grant, C.X.); and University of Minnesota (Ph.D. Dissertation
Fellowship, D.L.H.) for financial support.
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C.; Augeri, D. J.; Belli, B. A.; Bruncko, M.; Deckwerth, T. L.; Dinges,
J.; Hajduk, P. J.; Joseph, M. K.; Kitada, S.; Korsmeyer, S. J.; Kunzer, A.
R.; Letai, A.; Li, C.; Mitten, M. J.; Nettesheim, D. J.; Ng, S.; Nimmer,
P. M.; O’Connor, J. M.; Oleksijew, A.; Petros, A. M.; Reed, J. C.; Shen,
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ABBREVIATIONS USED
■
MDR, multidrug resistance; SAR, structure−activity relation-
ship; ABC, ATP-binding cassette; ER, endoplasmic reticulum;
IP3R, inositol triphosphate receptor; SERCA, sarco/endoplas-
mic reticulum Ca2+-ATPase; AML, acute myeloid leukemia;
ALL, acute lymphoblastic leukemia; CML, chronic myeloge-
nous leukemia; Ara-C, cytarabine; MX, mitoxantrone; ADR,
adriamycin; DNR, daunorubicin; HHT, homoharringtonine;
VLB, vinblastine; IAP, inhibitor of apoptosis; dCK, deoxy-
cytidine kinase; GI50, 50% growth inhibitory concentration;
SEM, standard error of the mean
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