Synthesis and anticancer activity of new coumarin-3-carboxylic acid derivatives as potential lactate transport inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115870

As an oncometabolite, lactate plays a very important role in tumor proliferation, metastasis, angiogenesis, immune escape and other tumor biological functions. Pharmacological inhibition of lactate transport has been viewed as a promising therapeutic strategy to target a range of human cancers. In this study, a series of new coumarin-3-carboxylic acid derivatives 5a-t and 9a-b were synthesized and evaluated as lactate transport inhibitors. Their cytotoxic activity has been tested against three cell lines high-expressing and low-expressing monocarboxylate transporter 1 (MCT1) which acts as the main carrier for lactate. Compound 5c-e, 5g-i and 5m-o showed significant cytotoxicity and good selectivity against Hela and HCT116 cell lines with high MCT1 expression. Notably, coumarin-3-hydrazide 5o, the lead molecule with the most potent cytotoxic activity, exhibited significant anti-proliferation and apoptosis induction effects. Further studies revealed that compound 5o decreased the expression level of target MCT1, and suppressed the energetic metabolism of Hela and HCT116 cells by remarkably reducing glucose consumption and lactate production. Additionally, compound 5o induced intracellular lactate accumulation and inhibited lactate uptake, which implied that it blocked lactate transport via MCT1. These results indicate a good start point for the development of lactate transport inhibitors as new anticancer agents.

Full text of DOI:10.1016/j.bmc.2020.115870

Journal Pre-proofs
Synthesis and anticancer activity of new coumarin-3-carboxylic acid deriva-
tives as potential lactatetransportinhibitors
Hong Ji, Yaling Tan, Nana Gan, Jing Zhang, Shannuo Li, Xuan Zheng,
Zhaohua Wang, Wei Yi
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https://doi.org/10.1016/j.bmc.2020.115870
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Please cite this article as: H. Ji, Y. Tan, N. Gan, J. Zhang, S. Li, X. Zheng, Z. Wang, W. Yi, Synthesis and
anticancer activity of new coumarin-3-carboxylic acid derivatives as potential lactatetransportinhibitors,
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Synthesis and anticancer activity of new coumarin-3-
carboxylic acid derivatives as potential
lactate transport inhibitors
Hong Ji1,*, Yaling Tan1, Nana Gan1, Jing Zhang1, Shannuo Li1, Xuan Zheng1,
Zhaohua Wang2,*, Wei Yi1,*
1Key
Laboratory
of
Molecular
Target
&
Clinical
Pharmacology
and the State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences
& the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, 511436, P. R.
China
2School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, 511436,
P. R. China
Corresponding author
Hong Ji, PhD
Associate Professor
Key Laboratory of Molecular Target
&
Clinical Pharmacology
School of
and the State Key Laboratory of Respiratory Disease,
Pharmaceutical Sciences & the Fifth Affiliated Hospital
Guangzhou Medical University
Xinzao Town, Panyu District, 511436, Guangzhou, China
E-mail: dljih@126.com
Zhaohua Wang, PhD
School of Basic Medical Sciences
Guangzhou Medical University
Xinzao Town, Panyu District, 511436, Guangzhou, China
E-mail: gy_wzhh@163.com
Wei Yi, PhD
Professor
Key Laboratory of Molecular Target
&
Clinical Pharmacology
School of
and the State Key Laboratory of Respiratory Disease,
Pharmaceutical Sciences & the Fifth Affiliated Hospital
Guangzhou Medical University
Xinzao Town, Panyu District, 511436, Guangzhou, China
E-mail: yiwei@gzhmu.edu.cn

Abstract: As an oncometabolite, lactate plays a very important role in tumor
proliferation, metastasis, angiogenesis, immune escape and other tumor
biological functions. Pharmacological inhibition of lactate transport has been
viewed as a promising therapeutic strategy to target a range of human
cancers. In this study, a series of new coumarin-3-carboxylic acid derivatives
5a-t and 9a-b were synthesized and evaluated as lactate transport inhibitors.
Their cytotoxic activity has been tested against three cell lines high-expressing
and low-expressing monocarboxylate transporter 1 (MCT1) which acts as the
main carrier for lactate. Compound 5c-e, 5g-i and 5m-o showed significant
cytotoxicity and good selectivity against Hela and HCT116 cell lines with high
MCT1 expression. Notably, coumarin-3-hydrazide 5o, the lead molecule with
the
most
potent
cytotoxic
activity,
exhibited significant
anti-
proliferation and apoptosis induction effects. Further studies revealed that
compound 5o decreased the expression level of target MCT1, and suppressed
the energetic metabolism of Hela and HCT116 cells by remarkably reducing
glucose consumption and lactate production. Additionally, compound 5o
induced intracellular lactate accumulation and inhibited lactate uptake, which
implied that it blocked lactate transport via MCT1. These results indicate a good
start point for the development of lactate transport inhibitors as new anticancer
agents.
Keywords: coumarin-3-carboxylic acid derivatives, synthesis, anticancer
activity, lactate transport inhibitors
1. Introduction
Normal cells, generally, metabolize glucose via oxidative phosphorylation
in the mitochondria where allows to oxidize pyruvate to carbon dioxide and
water in the presence of oxygen or by glycolysis whereby lactic acid is produced
in the cellular cytosol in hypoxic environments to maintain energy production [1].
However, the metabolic characteristics of tumor cells differ significantly from

those of normal cells. To fulfill tumorous bioenergetic and biosynthetic demands,
tumor cells are equipped with a high rate of glucose uptake and glycolytic
metabolism. Moreover, a metabolic switch to glycolysis from oxidative
phosphorylation attributing to respiration injury or mitochondrial dysfunction
which exhibits a defective tricarboxylic acid (TCA) cycle appears in glycolytic
tumor cells [2-4]. For these reasons, cancer cells prefer to utilize glycolysis to
support rapid proliferation even in aerobic condition, which is called the
“Warburg effect” in oncology [5]. A high rate of glycolytic metabolism inevitably
leads to an increasing rates of lactic acid production [6]. Lactic acid, the
glycolysis end products, actually need to be export to normalize intracellular pH
levels, causing extracellular acidosis [7]. Extracellular acidosis, as a common
feature of the tumor microenvironment, in line with lactate collaboratively
promotes tumor growth and metastasis, and consequently results in poor
prognosis and high mortality [8-11]. Additionally, more than a waste product,
lactate has been recently identified as a major energy fuel in tumors [12,13] and
has an important role as a potent signaling molecule in tumor metabolism [7]. In
this context, lactate transport and pH regulation currently are viewed as an
alternative therapeutic strategy in cancer treatment [14]
.
It has been indicated that lactate/pH homeostasis is maintained mainly via
membrane proteins monocarboxylate transporters (MCTs) [15,16] which belong
to the SLC16 gene family, presently composed by 14 members. MCTs are
responsible for the transport of monocarboxylates such as pyruvate, lactate,
and the ketone bodies [17,18]. Among the large family of MCTs, MCT1-4 are
described as H+/lactate symporters capable of bidirectional transport of lactic
acid across the plasma membrane. MCT1 has a broader distribution and higher
affinity for monocarboxylates. MCT2 although has a higher affinity for pyruvate
and lactate than MCT1, its expression is little in human tissues and primarily
restricted to those tissues that take up lactic acid for respiratory fuel or for
gluconeogenesis [19,20]. Expression of MCT3 is also confined to very specific
tissues in contrast to MCT1 [21,22]. It has been documented that MCT1 and

MCT4 are significantly overexpressed in an array of human malignant cancers
[23-27]. MCT1 is able to transport monocarboxylates into and out of cells,
whereas MCT4 predominates for exporting lactic acid [28,29]. MCT4 is quite
widely expressed especially in glycolytic/hypoxic tissues and has the lowest
[30]
affinity
for
pyruvate
and
lactate
.
Importantly,
the
downregulation of MCT1 inhibits tumor growth, invasion and metastasis, and
enhances chemotherapeutic efficacy in cancer[31,32]. Therefore, lactate
transport via MCT1 has been identified as a potential target for cancer therapy
[33]
.
Several MCT inhibitors have been identified, including the α-
[34]
cyanocinnamate
derivatives
,
stilbene
disulfonates
such
as
diisothiocyanostilbene disulfonate (DIDS), phloretin, bioflavenoids such as
quercetin and organomercurial reagents such as p-chloromercuribenzene
sulfonate[35]. However, none of these is specific for MCTs. Recently, some new
inhibitors have been developed that exhibit a high affinity for MCTs such as
[38]
AZD3965 [36], AR-C155858 [37], as well as pteridine diones and triones
.
AZD3965 developed by AstraZeneca has advanced into phase Ⅰ/Ⅱ clinical
trails [39]. However, they are dual MCT1/2 inhibitors [40]. MCT2 inhibition may
play a role in the potential off-target effects of current agents, which may result
in toxic side effects.
Coumarin derivatives present in various natural and synthetic compounds
[41-43]
are characterized by versatile pharmacological properties
such as
anticancer, anti-inflammation, antiviral, antimicrobial and anti-HIV effects. In
2013, Draoui and coworkers synthesized a series of monocarboxylate-
containing coumarins and identified them as a potential scaffold endowed with
inhibitory effects on lactate transport [44]. 7-alkylamino-3-carboxycoumarins
were then developed as potent specific MCT1 inhibitors which selectively
[45,46]
interfered with lactate fluxes in the lactate-rich tumor microenvironment
.
The lack of toxicity and a good ADME profile support the potential of this new
family of compounds to be developed as broad-spectrum anticancer agents [44-
46]
.

In order to study the influence of substituent effect on the lactate transport
inhibitory activity and expand the structure-activity relationship library of
carboxycoumarin derivatives, we focused on the coumarin-3-carboxylic acid
derivatives with changes to substituents at C-3 and C-7 positions of the
coumarin scaffold. The structural moieties of carboxamide, hydroxamic acid,
formylhydrazine and acylhydrazone at C-3 position of coumarin core were
designed. Herein, we report the synthesis of these coumarin-3-carboxylic acid
derivatives and their activity as lactate transport inhibitors that block the growth
and induce apoptosis of MCT1 high-expressing human cervix cancer cell Hela
and colorectal cancer cell HCT116.
2. Results and Discussion
2.1. Chemistry
O
O
O
O
O
O
H
piperidine, EtOH
rt, 3 h
+
O
O
R1
O
R1
OH
2
1
3
1a: R1 =
1b: R1 =
3a: R1 =
3b: R1 =
OH
OH
N
N
O
O
O
O
O
R2Br, K2CO3
O
DMF
rt, 4 h
R2O
O
HO
O
4
3a
4a: R2 =
4b: R2 =
4c: R2 =
4d: R2 =
O
O
O
Br
O
O
4e: R2 =
NO2
Scheme 1. Synthesis of compound 4a-e

O
R4
N
H
R4NH2, EtOH
reflux, 18-36 h
R3
O
O
O
5a-l
OH
N
H
NH2OH·HCl, NaOH
3b or 4a-e
EtOH
rt, 4-6 h
R3
O
O
O
5m-n
H2NNH2·H2O
NH2
N
H
EtOH
reflux, 0.5-1 h
R3
O
O
5o-t
R4=
OH
5a: R3 =
5j: R3 =
5k: R3 =
5l: R3 =
R4 =
R4 =
R4 =
O
N
O
O
R4=
OH
5b: R3 =
5c: R3 =
5d: R3 =
5e: R3 =
O
R4 =
N
OH
Br
R4 =
R4 =
O
O
OH
OH
N
5m: R3 =
5n: R3 =
5o: R3 =
5p: R3 =
5q: R3 =
5r: R3 =
O
N
NO2
R4 =
R4 =
R4 =
R4 =
5f: R3 =
5g: R3 =
5h: R3 =
5i: R3 =
OH
O
N
O
O
O
O
O
H
N
O
O
O
O
O
Br
H
N
Br
O
O
O
O
NO2
O
O
Br
5s: R3 =
5t: R3 =
H
N
O
O
O
NO2
NO2
Scheme 2. Synthesis of compound 5a-t
The target compounds 5a-t and 9a-b were synthesized as illustrated in
Scheme 1, 2 and 3. The key intermediates 3a-b was prepared by
Knoevenagel reaction of 4-substituted salicylaldehyde 1 with diethyl malonate
2 in the presence of piperidine in anhydrous ethanol. Treatment of 3a with
potassium carbonate and various bromides such as bromoethane, 1-
bromopropane, benzyl bromide, 3-bromobenzyl bromide, 4-nitrobenzyl

bromide in N,N-dimethylformamide afforded corresponding alkylated products
4a-e (Scheme 1). Thereafter, the resulted intermediates 3b and 4a-e were
refluxed with 2-aminoethanol or amine in ethanol to provide coumarin-3-
carboxamides 5a-f and 5j-l (Scheme 2), respectively. Reaction of esters 4c-e
and ethylenediamine unexpected gave products 5g-i instead of N-2-
aminoethyl-coumarin-3-carboxamides. In presence of sodium hydroxide,
refluxing of compound 3b or 4c with hydroxylamine hydrochloride in
ethanol gave coumarin-3-hydroxamic acids 5m and 5n, respectively.
Aminolysis reaction of esters 3b and 4a-e with hydrazine hydrate in ethanol
under reflux conditions led to coumarin-3-formylhydrazines 5o-t.
O
O
+
NC
NH2
R3
N
H
6
7
7a: R3 =
7b: R3 =
1,4-dioxane
reflux, 3h
S
N
O
O
O
O
N
NC
N
N
1,4-dioxane
reflux, 3h
H
H
N
R3
H
+
R3
R1
O
N
OH
9
8
1b
8a: R3 =
8b: R3 =
R3=
R3 =
9a: R1 =
9b: R1 =
N
S
S
N
N
N
Scheme 3. Syntheis of compound 9a-9b
Several attempts to transform formylhydrazines
5o into acylhydrazone derivatives were unfortunately failed. We then turned to
the synthetic route as depicted in Scheme 3. Target compounds 9a-b were
achieved in a two-step synthesis process. Intermediates 8a-b were obtained
starting from cyanoacetylhydrazine 6 and acetylated heterocycle 7 such as 2-
acetylthiophene and 3-acetylpyridine, followed by the condensation reaction of
intermediates 8a-b with 4-diethylamino salicylaldehyde 1b, leading to
acylhydrazones 9a-b (Scheme 3).

2.2. Expression of MCT1 in Two Different Human Cancer Cell Lines and a
Human Normal Cell Line
MCT1 expression was detected in two different human cancer cell lines
Hela (cervix cancer cell) and HCT116 (colorectal cancer cell) along with a
normal human colon mucosal epithelial cell line NCM460. Western blotting
results revealed that MCT1 is expressed at high level in both Hela and HCT116
cells, but low level in NCM460 cells (Figure 1A), which was also verified by the
transcriptional level of MCT1 in these cell lines. The expression of MCT1 in
NCM460 cells was used as a negative control. By real-time PCR, the
expression of MCT1 in Hela and HCT116 cells was about one hundred and
thirty-fold and one hundred and ninety-fold higher respectively, compared to
vehicle control (Figure 1B).
Figure 1. MCT1 is expressed in Hela and HCT116 cells. (A) Protein expression of MCT1
in Hela, HCT116 and NCM460 cells. (B) Relative mRNA levels of MCT1 in Hela, HCT116
and NCM460 cells were assessed by RT-PCR. Quantitative data were acquired as mean
± SEM using GAPDH as a housekeeping gene. Each experiment was done in triplicate.

2.3. Cytotoxic Activities against Two Human Cancer Cell Lines and a
Human Normal Cell Line
Twenty two newly synthesized compounds were evaluated against Hela,
HCT116 and NCM460 cells by means of MTT assay. Table 1 depicts the results
of their cytotoxic activity. All compounds showed no cytotoxicity to NCM460
cells with low MCT1 expression. Compounds 5c-e, 5g-i and 5m-o
showed potent cytotoxic effects against both Hela and HCT116 cells with IC50
values in the range of 3.36-68.07 μM. Compounds 5b, 5k, 5p and 9b exhibited
very low toxicity to HCT116 cells, but moderate to good activity against Hela
cells with IC50 value of 22.62 μM, 73.04 μM, 38.33 μM, and 70.01 μM,
respectively. Conversely, compound 5q had moderate toxicity for HCT116
cells, but low activity for Hela cells. Among the compounds, the most active is
compound 5o with an IC50 value of 5.48 μM for Hela cells and 3.36 μM for
HCT116 cells, exhibiting IC50 values lower than α-cyano-4-hidroxycinnamate
(CHC) (IC50 2.14 mM for Hela cells and 1.65 mM for HCT116 cells) and
quercetin (IC50 22.50 μM for Hela cells and 43.87 μM for HCT116 cells), which
were the representative MCT1 inhibitors and used as reference drugs. The
results of MTT assay in Hela and HCT116 cells using a set of top-performing
compounds are shown in Figure 2.
Table 1. Cytotoxic activities of compounds 5a-t and 9a-b in Hela and HCT116 cell lines
Compound
Cell line, IC50 (μM)
Compound
Cell line, IC50 (μM)
Hela
＞100
22.62
14.24
17.55
6.89
HCT116
＞100
＞100
18.72
30.81
33.36
＞100
15.91
11.57
68.07
＞100
＞100
＞100
Hela
35.87
31.76
5.48
HCT116
37.64
39.46
3.36
5a
5b
5c
5d
5e
5f
5m
5n
5o
5p
38.33
＞100
＞100
＞100
＞100
＞100
70.01
22.50
2140
＞100
69.81
＞100
＞100
＞100
＞100
＞100
43.87
1650
5q
＞100
12.13
5.52
5r
5g
5h
5i
5s
5t
13.93
＞100
73.04
＞100
9a
5j
9b
5k
5l
quercetin
CHC

Figure 2. MTT assays in Hela and HCT116 cell lines (n=3)
2.4. Effect of Compound 5o on Antiproliferation of Hela and HCT116 Cells
Depending on the results obtained from the cytotoxity test, the most potent
compound
on its antiproliferative effects against Hela and HCT116 cell lines.
Results from MTS assays showed a remarkable decline in
5o
was
selected
to
be
further
studied
the proliferative capacity of Hela cells after treated with compound 5o at IC50
concentration on day 6, and there was no statistical significance between
compound 5o and CHC (Figure 3A and 3B). For HCT116 cells, the suppression
effect was slightly different. Compound 5o exhibited a more prominent
antiproliferation effect. On day 6, the proliferative capacity of HCT116 cells was
significantly suppressed compared with the negative control group and

showed no significant difference as compared with quercetin (Figure 3C and
3D).
Figure 3. The MTS assays of compound 5o in Hela and HCT116 cell lines. The
proliferative capacities of Hela cells (A) and HCT116 cells (C) were detected by MTS
assays after treated with compound 5o, CHC or quercetin. The 6th day results were used
to analyze the variance. The 6th day of MTS assay results in Hela cells (B) and HCT116
cells (D). Data are the mean±SEM of triplicate values from three independent experiments.
*P<0.05, **P<0.01, compared to the denoted group. NS, no significant.
2.5. Effect of Compound 5o on Induction of Apoptosis in Hela and HCT116
Cells
Next, we checked the ability of compound 5o to induce apoptosis in Hela
and HCT116 cells. A commercial Apoptosis Detection Kit was used to evaluate
apoptosis by annexin V binding and propidium iodide (PI) staining. Control
tubes of unstained cells, cells stained with PI alone, and cells stained with
annexin V only were included for setting the flow cytometer compensation. The
apoptosis rate was calculated as the percentage of annexin V+ cells. Treatment
of Hela and HCT116 cells with compound 5o resulted in a significant
augmentation in the apoptotic cell population compared to untreated control

(Figure 4). For both Hela and HCT116 cells, CHC and quercetin caused more
apoptosis than compound 5o.
Figure 4. Compound 5o induced apoptosis in Hela (A) and HCT116 cells (B). The cells
were cultured with compound 5o, CHC or quercetin for 48 hours. FCM was used to analyze
apoptosis rate. The annexin V+ cell populations were calculated. Data presented are
representative of three independent experiments. *P<0.05, **p<0.01, NS, no significance.
2.6 Compound 5o Decreased Expression of MCT1 in Hela and HCT116
Cells
To further explore the mechanism of compound 5o for

lactate transport inhibition, western blotting was used to detect the levels of
MCT1 protein expression in Hela and HCT116 cells. As shown in Figure 5, after
treated with increasing concentration of compound 5o for 48 h, an apparent
decrease in the expression of MCT1 was observed in the both cancer cell lines.
The results suggest that compound 5o may exert lactate transport inhibitory
effect through down-regulation of MCT1 expression.
Figure 5. Expression of MCT1 protein in two cancer cell lines treated with compound
5o. Western blot analysis of MCT1 protein in Hela and HCT116 cells treated with different
concentrations of compound 5o (0.01, 0.1, 1, 10, 100 μM) for 48 h. Each experiment was
done in triplicate and a representative image is shown for each marker.
2.7. Compound 5o Impaired Energetic Metabolism of Hela and HCT116
Cells
The effect of compound 5o on metabolic disturbance resulted from lactate
transport inhibition was further evaluated. Quercetin and CHC were
used as references, which are known to inhibit lactate transport. The two cell
lines were treated with the correspondent IC50 values (Hela: compound 5o =
5.48 μM, CHC = 2.14 mM, quercetin = 22.50 μM; HCT116: compound 5o =
3.36 μM, CHC = 1.65 mM, quercetin = 43.87 μM) for 4, 8, 12, 24 hours, and the
intracellular and extracellular amounts of glucose and lactate were estimated
over time, respectively. The results are summarized in Figure 6, compound 5o
significantly decreased glucose consumption in Hela cells (Figure 6A), with
reduction already after 12 hours of incubation. A significant reduction in lactate
production was observed after 8 hours of incubation in Hela cells with no

difference between compound 5o, CHC and quercetin (Figure 6B). In HCT116
cells, the treatment with compound 5o or CHC and quercetin significantly
affected glucose consumption (Figure 6C). Regarding lactate production,
compound 5o, CHC or quercetin treatment showed a significant inhibitory effect
from the first 4 hours to the remaining incubation periods compared to the
negative control (Figure 6D).
Figure 6. Compound 5o reduced glucose consumption and lactate production in Hela (A
and B) and HCT116 cells (C and D). Cells were incubated with the IC50 values for
compound 5o, CHC or quercetin, and extracellular glucose and lactate were quantified
over-time (4, 8, 12 and 24 h). Values are expressed as mean ± SEM. Each experiment was
done in triplicate. Significant differences are presented as *p<0.05, **p<0.01, ns; Not
significant.
Further, the levels of intracellular glucose and lactate were evaluated.
Results showed that there was a significant decrease in the intracellular
glucose levels in compound 5o-treated Hela cells at 24 h (Figure 6A). CHC and
quercetin induced a rapid decrease after 4 hours of incubation. A significant
decrease was also observed in HCT116 cells after 4 h, whereas no significant
difference was detected between compound 5o and the negative control after
24 h (Figure 6C). In addition, incubation of Hela cells with compound 5o

substantially increased intracellular lactate at 8 h and 24 h, however, the effect
of compound 5o was smaller than positive controls (Figure 6B). A similar
increase in the levels of intracellular lactate provoked by compound 5o was also
manifest in HCT116 cells at 8 h (Figure 6D). Increases in the levels of
intracellular lactate and corresponding decreases in extracellular lactate
indicated that lactate transport had been blocked. The negative feedback upon
inhibition of lactate transport subsequently led to the reduction of glucose
consumption observed. Thus, the inhibitory effect of compound 5o disrupted
the energetic metabolism of Hela and HCT116 cells.
Figure 7. Effect of compound 5o on the levels of intracellular glucose and lactate in Hela
(A and B) and HCT116 cells (C and D). Cells were incubated with the IC50 values for
compound 5o, CHC and quercetin, and intracellular glucose and lactate were quantified
over time (4, 8, 12 and 24 h). Values are expressed as mean ± SEM. Each experiment was
done in triplicate. Significant differences are presented as *p<0.05, **p<0.01, ns; Not
significant.
2.8. Compound 5o Inhibited Lactate Uptake of Hela and HCT116 Cells
To get further insights on the profile of lactate flux inhibition by compound
5o, we examined the capacity of the molecule to interfere with lactate uptake.
As shown in Figure 8B, compound 5o blocked lactate uptake significantly in

HCT116 cells, compared with untreated cells. For Hela cells similar result was
observed at low concentration, but no effect at high concentration (Figure 8A).
CHC
exhibited
no inhibitory effect
on the
lactate
uptake
in
the micromolar range.
Figure 8. Compound 5o inhibited lactate uptake of Hela (A) and HCT116 cells (B). Cells
were incubated in lactate-containing medium with different concentrations of compound 5o,
CHC or quercetin (0.01, 0.1, 1, 10, 100 μM) for 24 h, and the remaining lactate
concentration in the extracellular medium were quantified. Values are expressed as mean
± SEM. Each experiment was done in triplicate. Significant differences are presented as
*p<0.05, **p<0.01, ns; Not significant.
2.9. Structure-activity Relationships (SARs)
According to the cytotoxic effects of the synthesized coumarin-3-carboxylic
acid derivatives 5a-t and 9a-b on the cancer cells Hela and HCT116 with MCT1
expression, a preliminary structure-activity relationship (SAR) was established.
Among the coumarin-3-carboxamides 5a-l, N-2-hydroxyethyl-coumarin-3-
carboxamides 5a-f and biscoumarin-3- formylethylenediamine 5g-i exhibited
higher activity compared to N-alkyl-coumarin-3-carboxamides 5j-l. Benzyloxy
group at the C-7 position of the coumarin scaffold is favored to the activity, and
the substituents on the benzene ring have no impact on the activity. The
biscoumarin derivatives 5g-i had higher activity than others. Hydroxamic acid
5m and 5n exhibited potent against the two cell lines regardless of the
difference of the substituents at C-7 position of the coumarin core. Except for
compound 5o, weak cytotoxicity against Hela and HCT116 cell lines was
observed for the majority of the coumarin-3-formylhydrazines, which indicated

diethylamino group at C-7 position drastically improved potency. In addition,
introduction of acylhydrazone moiety at C-3 position of the coumarin scaffold
led to a dramatic decrease of activity ( IC50 > 100 μM for compound 9a-b),
which implied the acylhydrazone moiety was unfavourable for cytotoxicity.
3. Conclusion
Owing to the importance of lactate transport in tumor metabolism, the
development of MCT1 inhibitors has been of immense importance in cancer
prognosis and therapy. Several types of MCT1 inhibitors have emerged in
recent years.,[36-38,44] However, few have been developed as specific
candidates for cancer treatment. Given the potency of coumarin-3-carboxylic
acids as MCT1 inhibitors, here we synthesized twenty-two 3-carboxycoumarin
derivatives classified into four different categories by using different reaction
conditions. Their structures were characterized by several spectroscopic
techniques. The cytotoxic activities of the compounds were screened using
Hela, HCT116 and NCM460 cell lines. For that, the expression of MCT1 in the
three cell lines was evaluated by western-blotting and RT-PCR assays. The
high expression of MCT1 was demonstrated in Hela and HCT116 cell lines. As
expected, most compounds which showed a non-toxic effect on NCM460 cells
with low MCT1 expression had different degrees of cytotoxicity against Hela
and HCT116 cell lines. Compounds 5c, 5g, 5h and 5o exhibited good
antiproliferative activity against both Hela and HCT116 cell lines. Compound
5o showing the highest activity were selected for further study. The
efficient antiproliferation and induction of apoptosis in Hela and HCT116 cell
lines by compound 5o were confirmed by MTS assay and flow cytometry.
Additionally, Compound 5o decreased the expression levels of MCT1, but its
mechanism of action remains to be further studied. Regarding energetic
metabolism, there was an overall decrease in both glucose consumption and
lactate production in Hela and HCT116 cells after treatment with compound 5o.

Significant increases in intracellular lactate and decreases in extracellular
lactate indicated the inhibitory effect of compound 5o on lactate transport. The
decreases in intracellular glucose and reduction of glucose consumption
observed are probably due to negative feedback upon inhibition of lactate
transport. Finally, the potential of compound 5o to act as MCT1 inhibitor was
further confirmed by its inhibition of lactate uptake. The pharmacological results
proved that the new coumarin-3-carboxylic acid derivatives are a privileged
scaffold for the design and discovery of novel reagents for anticancer treatment.
4. Materials and Methods
4.1. Synthesis
4.1.1. General Procedures
Melting points were determined using an X-6 microscope melting point
instrument (Bjfuka, China) and are uncorrected. NMR spectra were obtained
using 400 MHz JNM-ECZ400S/L1 spectrometers (Varian INOVA, America)
with tetramethylsilane (TMS) as an internal standard. Infrared spectra were
obtained using a TENSORⅡspectrometer (Bruker, German). HR-MS spectra
were obtained on Finnigan LCQ and Micromass Auto Spec Ultima-Tof
spectrometer (Thermo, America). The progress of the reactions was monitored
by thin-layer chromatography (TLC) on 200 mm×200 mm aluminum sheets
(silica gel 60, HF-254, Hailang) with a thickness of 0.2-0.25 mm, using
ultraviolet light irradiation. Unless otherwise noted, all solvents and reagents
were used without further purification.
4.1.2. General Procedure for the Synthesis of Ethyl Coumarin-3-formate
Derivatives 3a-b [47,48]
Compounds 3a-b were prepared by the reaction between 4-substituted
salicylaldehyde 1 (1.00 eq., 15.20 mmol) and diethyl malonate (5.0 mL, 30.40
mmol). 4-substituted salicylaldehyde 1 was dissolved in anhydrous ethanol
before the diethyl malonate was slowly added to the solution. Piperidine (1.0
mL) was added dropwise to catalyze the reaction. The reaction mixture was

stirred at room temperature for 3h. 10% HCl was added into the reaction
mixture to adjust pH above 7 and the resulting solid was filtered and washed
with cold ethanol and recrystallized from ethanol, leading to the pure derivatives
3a-b as yellow solid.
4.1.3. General Procedure for the Synthesis of Ethyl Coumarin-3-formate
Derivatives 4a-e [49,50]
Compounds 4a-e were prepared by the reaction between compound 3a
(1.17 g, 5.00 mmol) and the appropriate bromides (1.40 eq., 7.00 mmol).
Compound 3a was dissolved in anhydrous N,N-Dimethylformamide (10 mL),
followed by the addition of the bromide and potassium carbonate (829 mg, 6.00
mmol). The reaction mixture was stirred at room temperature for 4h, then
poured into water (20 mL). The resulting solid was filtered and washed with
water, leading to the derivatives 4a-e.
4.1.4. General Procedure for the Synthesis of Coumarin-3-carboxamides
5a-l
Coumarin-3-carboxamides 5a-l were prepared by the reaction between
compounds 3b and 4a-e (1.00 eq.,1.00 mmol) and the appropriate primary
amine (1.20 eq., 1.20 mmol) in ethanol (10.0 mL). The reaction mixture was
heated under reflux for 18-36 h. The solvent was then evaporated under
reduced pressure, followed by the addition of water (10.0 mL) and 10% HCl to
adjust pH to 7-8 before liquid extraction with ethyl acetate. The organic layers
were merged and dried over anhydrous sodium sulphate, then filtered and
concentrated under reduced pressure. The resulting crude was purified via
column chromatography on silica gel.
7-Ethoxy-N-(2-hydroxyethyl)-2-oxo-2H-chromene-3-carboxamide (5a).
Yellow solid; 74% yield; m.p: 111-113 ℃; IR (KBr) νmax (cm-1) 3150, 1651, 1610;
1H NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 7.02 (d, J = 8.3 Hz, 1H), 6.28-6.33
(m, 2H), 4.01 (q, J = 7.0 Hz, 2H), 3.85 (dd, J = 5.7, 4.6 Hz, 2H), 3.64 (t, J = 5.7,
13
2H), 1.40 (t, J = 7.0 Hz, 3H); C NMR (100 MHz, CDCl3) δ 167.98, 165.42,
163.91, 133.17, 111.86, 107.12, 102.05, 63.68, 62.01, 59.29, 14.76; HR-MS

(ESI): Calcd for C14H17NO5 (M + H+): 278.1028; Found : 278.1032.
N-(2-hydroxyethyl)-2-oxo-7-propoxy-2H-chromene-3-carboxamide
(5b). Yellow solid; 70% yield; m.p: 90-92 ℃. IR (KBr) νmax (cm-1) 3274, 1634,
1
1608; H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 6.99-7.05 (m, 1H), 6.31 (s,
2H), 3.89 (d, J = 6.6 Hz, 2H), 3.85 (t, J = 4.9 Hz, 2H), 3.62 (d, J =4.9 Hz, 2H),
13
1.79 (m, 2H), 1.02 (t, J = 7.5 Hz, 3H); C NMR (100 MHz, CDCl3) δ 168.19,
165.37, 133.17 , 111.79, 107.14, 102.11, 69.66, 61.98, 59.18, 22.51, 10.58;
HR-MS (ESI): Calcd for C15H18NO5 (M + H+): 292.1185; Found: 292.1184.
7-(Benzyloxy)-N-(2-hydroxyethyl)-2-oxo-2H-chromene-3-
carboxamide (5c). Yellow solid; 73% yield. m.p:117.3-119.4 ℃. IR (KBr) νmax
(cm-1) 3164, 2873, 1640, 1607; 1H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.30-
7.46 (m, 5H), 7.03 (d, J = 8.5 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 6.37- 6.41 (m,
1H), 5.06 (s, 2H), 3.86(t, J = 5.0 Hz, 2H), 3.64 (t, J = 5.0 Hz, 2H);13C NMR (100
MHz, CDCl3) δ 167.59, 165.53, 163.50, 136.59, 133.22, 128.75, 128.23, 127.68,
112.19, 107.28, 102.57, 70.04, 62.07, 59.52; HR-MS (ESI): Calcd for
C19H18NO5 (M + H+): 340.1185; Found: 340.1182.
7-[(3-Bromobenzyl)oxy]-N-(2-hydroxyethyl)-2-oxo-2H-chromene-3-
carboxamide (5d). Yellow solid; 65% yield; m.p:111-113 ℃. IR (KBr) νmax (cm-1)
1
3264, 2998, 1638, 1621; H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 7.58 (s,
1H), 7.43-7.49 (m, 1H), 7.34 (d, J = 7.7 Hz, 1H), 7.25 (m, 1H), 7.05-7.11 (m,
1H), 6.42 (d, J = 7.5 Hz, 2H), 5.03 (s, 2H), 3.88 (t, J = 4.8 Hz, 2H), 3.67 (t, J =
13
4.8 Hz, 2H) C NMR (100 MHz, CDCl3) δ 166.81, 165.69, 162.94, 138.93,
133.20, 131.30, 130.49, 130.32, 126.02, 122.86, 112.54, 107.19, 102.50, 69.11,
62.18, 59.93; HR-MS (ESI): Calcd for C19H17BrNO5 (M + H+): 418.0290; Found:
418.0287.
N-(2-hydroxyethyl)-7-[(4-nitrobenzyl)oxy]-2-oxo-2H-chromene-3-
carboxamide (5e). Yellow solid; 83% yield; m.p:125-126 ℃. IR (KBr) νmax (cm-1)
3531, 1620, 1512, 1340; 1H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 8.7 Hz, 3H),
7.60 (d, J = 8.6 Hz, 2H), 7.15 (d, J = 9.1 Hz, 1H), 6.46 (d, J = 7.3 Hz, 2H), 5.18
13
(s, 2H), 3.90 (t, J = 4.9 Hz, 2H), 3.70 (t, J = 4.9 Hz 2H); C NMR (100 MHz,

CDCl3) δ 166.07, 165.81, 162.31, 147.75, 144.01, 133.21, 127.76, 124.01,
112.88, 107.03, 102.45, 68.66, 62.24, 60.26; HR-MS (ESI): Calcd for
C19H17N2O7 (M + H+): 385.1036; Found: 385.1032.
7-(Diethylamino)-N-(2-hydroxyethyl)-2-oxo-2H-chromene-3-
carboxamide (5f) [51]. Yellow solid; 71% Yield; m.p:121-123 ℃. IR (KBr) νmax
(cm-1) 3386, 3320, 1693,1622; 1H NMR (400 MHz, CDCl3) δ 9.17 (s, 1H), 8.71
(s, 1H), 7.44 (d, J = 9.0 Hz, 1H), 6.67 (dd, J = 9.0, 2.5 Hz, 1H), 6.51 (d, J = 2.3
Hz, 1H), 3.84 (t, J = 6.2 Hz, 2H), 3.63 (t, J = 6.2 Hz, 2H), 3.47 (q, J = 7.1 Hz,
4H), 1.26 (t, J = 7.1 Hz, 6 H); 13C NMR (100 MHz, CDCl3) δ 165.02, 162.75,
157.76, 152.75, 148.37, 131.28, 110.08, 109.70, 108.36, 96.60, 63.23, 45.14,
43.20, 12.45; HR-MS (ESI): Calcd for C16H20N2O4 (M + H+): 305.1501; Found:
305.1498.
N,N'-(ethane-1,2-diyl)bis(7-(benzyloxy)-2-oxo-2H-chromene-3-
carboxamide) (5g). Yellow solid; 66% yield; m.p:148-150 ℃. IR (KBr) νmax (cm-
1) 1631, 1580; 1H NMR (400 MHz, CDCl3) δ 8.20 (s, 2H), 7.31-7.45 (m, 10H),
7.09 (d, J = 8.6 Hz, 2H), 6.50 (d, J = 2.3 Hz, 2H), 6.46 (dd, J = 8.5, 2.4 Hz, 2H),
5.05 (s, 4H), 3.84 (s, 4H);13C NMR (100 MHz, CDCl3) δ 165.54, 164.77, 162.77,
136.56, 132.89, 128.74, 128.21, 127.63, 112.64, 107.18, 102.26, 70.10, 58.91;
HR-MS (ESI): Calcd for C36H29N2O8 (M + H+): 617.1924; Found: 617.1919.
N,N'-(ethane-1,2-diyl)bis(7-((3-bromobenzyl)oxy)-2-oxo-2H-
chromene-3-carboxamide) (5h). Yellow solid; 68% yield; m.p:137-138 ℃. IR
(KBr) νmax (cm-1) 3064, 2997, 1624, 1517; 1H NMR (400 MHz, CDCl3) δ 8.20 (s,
2H), 7.57 (t, J = 1.6 Hz, 2H), 7.42-7.48 (m, 2H), 7.32 (m, 2H), 7.25 (m, 2H), 7.10
(d, J = 8.5 Hz, 2H), 6.45 (m, 4H), 5.01 (s, 4H), 3.85 (s, 4H);13C NMR (100 MHz,
CDCl3) δ 165.54, 164.81, 162.41, 138.91, 132.98, 131.28, 130.46, 130.31,
125.98, 122.84, 112.82, 107.11, 102.26, 69.14, 58.88; HR-MS (ESI): Calcd for
C36H27Br2N2O8 (M + H+): 773.0134; Found: 773.0138.
N,N'-(ethane-1,2-diyl)bis(7-((4-nitrobenzyl)oxy)-2-oxo-2H-chromene-
3-carboxamide) (5i). Yellow solid; 70% yield; m.p:158-160 ℃. IR (KBr) νmax
1
(cm-1) 3289, 3089, 1625, 1518, 1288, 1221; H NMR (400 MHz, DMSO-d6) δ

8.43 (s, 2H), 7.50 (s, 2H), 7.38-7.44 (m, 6H), 7.28 (d, J = 8.6 Hz, 2H), 6.46 (dd,
J = 8.6, 2.4 Hz, 2H), 6.41 (d, J = 2.4 Hz, 2H), 5.13 (s, 4H), 3.82 (s, 4H). 13C
NMR (100 MHz, DMSO-d6) δ 165.84 , 165.19 , 162.00 , 139.31 , 133.26 ,
133.11 , 130.41 , 127.83 , 127.32 , 126.20 , 112.25 , 106.43 , 102.00 , 68.29 ,
57.25; HR-MS (ESI): Calcd for C36H27N4O12 (M + H+): 707.1625; Found:
707.1627.
7-(benzyloxy)-2-oxo-N-phenyl-2H-chromene-3-carboxamide
(5j).
Yellow solid; 70% yield; m.p: 213-215 ℃. IR (KBr) νmax (cm-1) 3352, 3047, 1700,
1620; 1H NMR (400 MHz, CDCl3) δ 8.50 (s, 1H), 7.31-7.43 (m, 7H), 7.23 (dd, J
= 5.5, 4.3 Hz, 4H), 6.51-6.59 (m, 2H), 5.07 (s, 2H); 13C NMR (100 MHz, CDCl3)
δ 164.05, 163.20, 161.60, 148.45, 136.39, 133.69, 129.50, 128.79, 128.31,
127.68, 126.55, 121.13, 113.41, 107.86, 102.19, 70.21; HR-MS (ESI): Calcd
for C23H18NO4 (M + H+): 372.1236; Found: 372.1124.
7-(Diethylamino)-N-isopropyl-2-oxo-2H-chromene-3-carboxamide
(5k). Yellow solid; 70% yield; m.p:137-139 ℃. IR (KBr) νmax (cm-1) 3330, 1690,
1646; 1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 7.40 (d, J = 8.9 Hz, 1H), 6.62
(dd, J = 8.9, 2.5 Hz, 1H), 4.22 (m, 1H), 3.43 (q, J = 7.1 Hz, 4H), 1.20-1.26 (m,
13
12H); C NMR (100 MHz, CDCl3) δ 162.82, 162.22, 157.66, 152.50, 148.02,
131.12, 109.95, 108.50, 96.64, 45.15, 41.58, 22.88, 12.53; HR-MS (ESI): Calcd
for C17H23N2O3 (M + H+): 303.1709; Found: 303.1711.
N-cyclopropyl-7-(diethylamino)-2-oxo-2H-chromene-3-carboxamide
(5l). Yellow solid; 65% yield; m.p:141-142 ℃. IR (KBr) νmax (cm-1) 3321, 1692
1
and 1651; H NMR (400 MHz, CDCl3 ) δ 8.77 (s, 1H), 8.68 (s, 1H), 7.38-7.42
(m, 1H), 6.62 (dd, J = 9.0, 2.5 Hz, 1H), 6.46 (d, J = 2.4 Hz, 1H), 3.42 (q, J = 7.1
Hz, 4H), 2.91 (tt, J = 7.2, 3.8 Hz, 1H), 1.21 (t, J = 7.1 Hz, 6H), 0.81 (td, J = 7.0,
5.3 Hz, 2H), 0.61 (m, 2H);13C NMR (100 MHz, CDCl3) δ 164.53, 162.78, 157.65,
152.56, 147.93, 131.19, 110.19, 109.99, 108.40, 96.57, 45.13, 22.78, 12.48,
6.64; HR-MS (ESI): Calcd for C17H21N2O3 (M + H+): 301.1552; Found: 301.1556.
4.1.5. General Procedure for the Synthesis of Coumarin-3-hydroxamic
Acids 5m-n

An ethanol solution of compound 3b or 4c (0.46 mmol; 0.46 M) was added
to an aqueous solution of sodium hydroxide (1.85 mmol; 74 mg) and
hydroxylamine hydrochloride (0.93 mmol; 46.50 M). The mixture was stirred at
room temperature for 4-6 h. 5% HCl was added to the reaction solution to adjust
pH to 5.5, and then extracted with ethyl acetate (30 mL×3). The organic phases
were combined, dried with anhydrous sodium sulphate before concentrated
under vacuum. The crude product was purified by column chromatography on
silica gel.
7-(Diethylamino)-N-hydroxy-2-oxo-2H-chromene-3-carboxamide (5m).
White solid; 69% yield; m.p: 100-101 ℃. IR (KBr) νmax (cm-1) 3430, 3386, 1633,
576; 1H NMR (400 MHz, CDCl3 ) δ 9.84 (s, 1H), 8.09 (s, 1H), 6.95 (t, J = 10.8
Hz, 2H), 6.23 (m, 2H), 3.36 (q, J = 7.1 Hz, 4H), 1.17 (t, J = 7.1 Hz, 6H); 13C
NMR (100 MHz, CDCl3) δ 159.06, 153.07, 150.35, 131.98, 104.90, 103.87,
98.33, 44.58, 12.73; HR-MS (ESI): Calcd for C14H17N2O4 (M + H+): 277.1188;
Found: 277.1190.
7-(Benzyloxy)-N-hydroxy-2-oxo-2H-chromene-3-carboxamide
(5n).
White solid; 63% yield; m.p: 78-80 ℃. IR (KBr) νmax (cm-1) 3422, 1635, 1554;
1H NMR (400 MHz, CDCl3) δ 10.09 (s, 1H), 8.16 (s, 1H), 7.51 (s, 1H), 7.33-7.45
(m, 5H), 7.08 (d, J = 8.4 Hz, 1H), 6.61 (d, J = 2.3 Hz, 1H), 6.57 (m, 1H), 5.07
(s, 2H); 13C NMR (100 MHz, CDCl3) δ 161.46, 159.01, 152.76, 136.49, 131.90,
128.76, 128.26, 127.64, 110.09, 107.72, 102.48, 70.18; HR-MS (ESI): Calcd
for C17H14NO5 (M + H+): 312.0872; Found: 312.0867.
4.1.6. General Procedure for the Synthesis of Coumarin-3-
formylhydrazines 5o-t
Compounds 5o-t were synthesized from 3b and 4a-e (1.00 eq.; 1.00 mmol)
and hydrazine hydrate (40 mmol; 2.0 mL) in ethanol (5 mL) under reflux for 0.5-
1 h. The reaction solution was evaporated under reduced pressure, then water
(10 mL) was added before liquid extraction with ethyl acetate. The organic
phases were combined and dried with anhydrous anhydrous sodium sulphate,
then filtered and concentrated under vacuum. The resulting crude product was

purified by column chromatography on silica gel.
[52]
7-(Diethylamino)-2-oxo-2H-chromene-3-carbohydrazide
(5o)
.
Orange solid; 69% yield; m.p:188-190 ℃; IR (KBr) νmax (cm-1) 3429, 1630, 1592;
1H NMR (400 MHz, CDCl3) δ 9.71 (s, 1H), 8.67 (s, 1H), 7.42 (d, J = 9.0 Hz, 1H),
6.65 (dd, J = 9.0, 2.4 Hz, 1H), 6.49 (m, 1H), 4.13 (d, J = 3.7 Hz, 2H), 3.45 (q, J
= 7.1 Hz, 4H), 1.23 (t, J = 7.1 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 163.99,
162.22, 157.71, 152.77, 148.14, 131.25, 110.11, 109.12, 108.32 , 96.67, 45.21,
12.51; HR-MS (ESI): Calcd for C14H18N3O3 (M + H+): 276.1348; Found:
276.1352.
7-Ethoxy-2-oxo-2H-chromene-3-carbohydrazide (5p). Yellow solid; 62%
1
yield; m.p:195-197 ℃; IR (KBr) νmax (cm-1) 3423, 3076, 1622, 1574; H NMR
(400 MHz, CDCl3) δ 11.73 (s, 1H), 8.56 (s, 1H), 7.20 (m, 1H), 6.48-6.53 (m,
2H), 4.07 (q, J = 7.0 Hz, 2H), 1.43 (t, J = 7.0 Hz, 3H).13C NMR (100 MHz, CDCl3)
δ 163.36, 162.89, 161.85, 133.60, 111.10, 108.04, 101.75, 63.90, 14.81; HR-
MS (ESI): Calcd for C12H13N2O4 (M + H+): 249.0875; Found: 249.0874.
2-Oxo-7-propoxy-2H-chromene-3-carbohydrazide (5q). Yellow solid;
1
64% yield; m.p:217-218 ℃. IR (KBr) νmax (cm-1) 3425, 3074, 1619, 1573; H
NMR (400 MHz, CDCl3 ) δ 7.83 (s, 1H), 6.98 (d, J = 8.5 Hz, 1H), 6.47 (d, J =
2.4 Hz, 1H), 6.42 (dd, J = 8.5, 2.4 Hz, 1H), 5.27 (s, 2H), 3.91 (t, J = 6.6 Hz, 2H),
1.79 (q, J = 6.8 Hz, 2H), 1.02 (t, J = 7.5 Hz, 3H). 13C NMR (100 MHz, DMSO-
d6) δ 159.39, 158.28, 142.87, 129.34, 112.90, 105.85, 101.44, 68.90, 22.00,
10.40; HR-MS (ESI): Calcd for C13H15N2O4 (M + H+): 263.1032; Found:
263.1035.
7-(Benzyloxy)-2-oxo-2H-chromene-3-carbohydrazide (5r). Yellow solid;
1
53% yield; m.p:178-179 ℃. IR (KBr) νmax (cm-1) 3298, 3032, 1875, 1620; H
NMR (400 MHz, CDCl3 ) δ 11.73 (s, 1H), 8.58 (s, 1H), 7.32-7.45 (m, 5H), 7.23
(d, J = 8.2 Hz, 1H), 6.56-6.64 (m, 2H), 5.10 (s, 2H);13C NMR (100 MHz, DMSO-
d6) δ 166.32, 162.42, 162.09, 160.61, 156.27, 136.57, 132.74, 128.63, 128.50,
127.79, 114.99, 111.60, 107.78, 101.87, 69.48; HR-MS (ESI): Calcd for
C17H15N2O4 (M + H+): 311.1032; Found: 311.1031.

7-[(3-Bromobenzyl)oxy]-2-oxo-2H-chromene-3-carbohydrazide (5s).
Yellow solid; 64% yield; m.p: 208-210 ℃. IR (KBr) νmax (cm-1) 3328, 3180, 1624,
1
1570; H NMR (400 MHz, CDCl3) δ 11.26 (s, 1H), 7.79 (s, 1H), 7.54 (s, 1H),
7.41 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 7.4 Hz, 1H), 7.18-7.24 (m, 2H), 6.97 (d, J
= 8.5 Hz, 1H), 6.50 (d, J = 2.5 Hz, 1H), 6.45 (dd, J = 8.5, 2.5 Hz, 1H), 5.26 (s,
1H), 4.98 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 163.66, 163.21, 162.69, 161.56,
136.39, 133.69, 128.80, 128.33, 127.67, 122.57, 122.48, 116.38, 116.15,
113.36, 107.93, 102.21; HR-MS (ESI): Calcd for C17H14BrN2O4 (M + H+):
389.0137; Found: 389.0139.
7-[(4-Nitrobenzyl)oxy]-2-oxo-2H-chromene-3-carbohydrazide
(5t).
Yellow solid; 58% yield; m.p: 276-278 ℃; IR (KBr) νmax (cm-1) 3402, 3283, 1606,
1576; 1H NMR (400 MHz, DMSO-d6) δ 8.24 (d, J = 8.8 Hz, 2H), 7.88 (s, 1H),
7.69 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.4 Hz, 1H), 6.66 (s, 2H), 6.46-6.54 (m,
2H), 5.26 (s, 2H).13C NMR (100 MHz, CDCl3) δ 163.13, 162.99, 161.86, 136.38,
133.69, 128.82, 128.36, 127.67, 124.61, 124.12, 123.62, 111.47, 108.27,
102.34, 70.31; HR-MS (ESI): Calcd for C17H14N3O6 (M + H+): 356.0883; Found:
356.0880.
4.1.7. General Procedure for the Synthesis of Acetohydrazide 8a-b [53,54]
Acetohydrazide 8a-b were synthesized from cyanoacetylhydrazine 6 (0.01
mmol, 990 mg) and acetylated heterocycle 7 (1.00 eq.; 0.01 mmol) in 1,4-
dioxane (30 mL) under reflux for 3h. The reaction mixture was then poured into
ice-water (20 mL) and the resulting solid was filtered and washed with ethyl
acetate (2.0 mL), leading to the pure derivatives 8a-b.
[53]
2-Cyano-N'-[1-(thiophen-2-yl)ethylidene]acetohydrazide
(8a)
.
Yellow crystals; 72% yield; 1H NMR (400 MHz, DMSO-d6) δ 11.04 (s, 1H), 7.57
(s, 1H), 7.45 (d, J = 3.7 Hz, 1H), 7.07 (d, J = 3.7 Hz, 1H), 4.09 (s, 2H), 2.27 (s,
3H); 13C NMR (100 MHz, DMSO-d6) δ 165.28, 145.78, 142.94, 128.63, 127.80,
127.64, 116.03, 24.63, 14.31.
2-Cyano-N'-[1-(pyridin-3-yl)ethylidene]acetohydrazide (8b) [54]. White
crystals; 74% yield; 1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 8.93- 9.02

(m, 1H), 8.58 (dd, J = 4.7, 1.4 Hz, 1H), 8.16 (dt, J = 7.9, 1.9 Hz, 1H), 7.43 (dd,
J = 7.9, 4.8 Hz, 1H), 4.26 (s, 2H), 2.29 (d, J = 11.7 Hz, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 166.11, 150.04, 147.58, 147.24, 133.78, 133.42, 123.50, 116.31,
25.05, 13.83.
4.1.8. General Procedure for the Synthesis of Coumarin-3-acylhydrazones
9a-b
Compounds 9a-b were prepared by the reaction between compounds 8a-
b (1.00 eq.; 0.50 mmol) and 4-diethylamino salicylaldehyde 1b (193 mg; 1.00
mmol) in 1,4-dioxane (20 mL). The reaction mixture was heated for 3 h under
reflux.The reaction solution was evaporated under reduced pressure, then
water (10 mL) was added before liquid extraction with ethyl acetate. The
organic layers were merged and dried over anhydrous sodium sulphate, then
filtered and concentrated under vacuum. The resulting crude product was
purified by column chromatography on silica gel.
7-(Diethylamino)-2-oxo-N'-[1-(thiophen-2-yl)ethylidene]-2H-
chromene-3-carbohydrazide (9a). Yellow powder; 57% yield; m.p: 217-219 ℃.
1
IR (KBr) νmax (cm-1) 3316, 1668, 1444; H NMR (400 MHz, CDCl3) δ 8.50 (s,
1H), 7.31-7.36 (m, 4H), 7.00-7.03 (m, 1H), 6.51 (d, J = 11.2 Hz, 1H), 6.33 (s,
1H), 3.43 (d, J = 7.1 Hz, 4H), 2.36 (s, 3H), 1.23 (t, J = 7.1 Hz, 6H);13C NMR
(100 MHz, CDCl3) δ 160.11, 159.32, 156.58, 151.90, 148.30, 143.92, 143.36,
131.05, 128.21, 127.18, 126.84, 112.76, 108.57, 108.17, 96.35, 45.03, 15.54,
12.66; HR-MS (ESI): Calcd for C20H22N3O3S (M + H+): 384.1382; Found:
384.1379.
7-(Diethylamino)-2-oxo-N'-[1-(pyridin-3-yl)ethylidene]-2H-chromene-
3-carbohydrazide (9b). Yellow powder; 59% yield; m.p: 207-209 ℃; IR (KBr)
νmax (cm-1) 3318, 1669; 1H NMR (400 MHz, CDCl3) δ 8.99 (d, J = 2.3 Hz, 1H),
8.59 (d, J = 4.8, 1.6 Hz, 1H), 8.52 (s, 1H), 8.31 (d, J = 8.1, 2.0 Hz, 1H), 7.38 (s,
1H), 7.31 (s, 1H), 6.51 (m, 1H), 6.32 (s, 1H), 3.43 (q, J = 7.1 Hz, 4H), 2.38 (s,
13
3H), 1.22 (t, J = 7.2 Hz, 6H); C NMR (100 MHz, CDCl3) δ 160.74, 159.33,
156.66, 152.03, 150.20, 149.65, 148.05, 143.76, 134.42, 134.18, 131.15,

123.37, 112.39, 108.64, 108.09, 96.32, 45.05, 14.93, 12.65; HR-MS (ESI):
Calcd for C21H23N4O3 (M + H+): 379.1770; Found: 379.1774.
4.2. Biological Assays
4.2.1. Cell Lines and Culture Conditions
Human Cervix cancer cells (Hela) and colorectal cancer cells (HCT116)
and breast cancer cell (MDA-MB-231)，which were kindly provided by ATCC,
were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented
with 10% FCS, 100U/ml penicillin, 100ug/ml streptomycin under standard
conditions at 37℃ and 5% CO2 according to manufacturer’s instructions.
4.2.2. Western Blotting
After different treatment as indicated, cells were washed twice with PBS
and subsequently lysed in radioimmunoprecipitation assay (RIPA) buffer with 1%
PMSF. Protein concentration was measured using the bicinchonic acid (BCA)
Protein Assay Kit (Beyotime), after the removal of the cell debris by
centrifugation (12,000g, 5 min). Equal amounts of protein samples (50 μg) were
boiled for 10 min in sample buffer, separated by 10% SDS-PAGE and
transferred to a polyvinylidene fluoride (PVDF) membrane. The nonspecific
binding was blocked with TBS containing 0.1% Tween 20, 2% BSA, and 5%
nonfat dry milk for 1 h at room temperature. The membranes were then
immunoblotted with primary antibody, rabbit anti-human MCT1( Abcam, diluted
1:1000) and mouse anti-human tubulin (Beyotime, diluted 1:1000) and
incubated overnight in 4 ℃, respectively. The next day, after washed in TBS
containing 0.1% (vol/vol) Tween-20, the bands were incubated with a 1:5000
dilution of horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit
IgG antibodies (Invitrogen) at room temperature for 1 h, respectively.
Visualization was developed using the ECL plus Western blotting detection kit
(Amersham, Helsinki, Finland).
4.2.3. RT-PCR
Total RNA of HCT116 and Hela cells were extracted using trizol agents

(Takara) following the manufacturer’s instructions. Utilizing random primers and
cDNA Reverse Transcription Kit (Takara), afterwards, reverse-transcribed
cDNA was generated from the mRNA template. Each 2-μL cDNA sample was
subjected to real-time PCR, which uses adequately SYBR Premix Ex Tag Ⅱ
(Takara) and the QuantStudio 5 PCR Detection System (Life Technologies). To
compare the MCT1 mRNA levels in different cell lines, the following primers
were used:
MCT1 forward:5’- GGTGGAGGTCCTATCAGCAGT-3’,
reverse: 5’-CAGAAAGAAGCTGCAATCAAGC-3’;
GAPDH, forward: 5´-GCACCGTCAAGGCTGAGAAC-3´,
reverse: 5´-TGGTGAAGACGCCAGTGGA-3´.
The conditions for real-time PCR were as follows: 95 ℃ for 30 s; 40 cycles
at 95 ℃ for 5 s; and 60 ℃ for 30 s. Then dissociation steps were carried out at
95 ℃ for 15 s; 60 ℃ for 1 min; and 95 ℃ for 15 s.
4.2.4. MTT Assay
Hela (2000 cells/well) and HCT116(3000 cells/well) cells were seeded in
96-well microtiter plates respectively overnight incubation at 37℃ in a 5% CO2
humidified incubator. CHC (Tokyo chemistry Industry) and quercetin (Aladdin),
as well as self-synthesized coumarin derivatives were dissolved in dimethyl
sulfoxide (DMSO; MP Biomedicals, LLC) as reserve solution, and stored at -
20℃. The compounds were then subsequently diluted in DMEM and added 10
ul into per well with varying final concentrations (50, 25, 12.5, 6.25, 3.125,
1.5625 and 0.78125 μM) for 72 h exposure time. The cells were incubated with
10 ul of 5 mg/mL MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-
tetrazolium bromide, Thiazolyl Blue Tetrazolium Bromide, GENVIEW) solution
for 4 h at 37℃ in a 5% CO2 incubator. The supernatant was discarded, and the
media were then replaced with 100 ul of dimethyl sulfoxide to solubilize
formazan crystals in the wells. The absorbance was measured at 540/655 nm
using Thermo Spectrophotometer. The experiments were performed in
triplicate.

4.2.5. Proliferation (MTS)
Cell viability was evaluated by measuring the absorbance of MTS [3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium] (Promega). Hela (2000/well) or HCT116 (3000/well) cells were
seeded in each well of a 96-well plate and cultured overnight, followed by the
compound 5o, CHC and quercetin as indicated in the individual figure legends.
These compounds were added with the IC50 concentration calculated previous
ly. The viable cells were counted and evaluated by MTS assay according to the
manufacturer’s protocol. The absorbance value of each experimental well was
read at 490 nm on a spectrophotometer and normalized by subtracting the
mean of blank-well values.
4.2.6. Apoptosis
Hela and HCT116 cells were seeded at 2×105 cells/well in 24-well plates
overnight, and were treated by compound 5o, CHC and quercetin for 48 hours,
respectively. The cells were harvested and resuspended in 100 µl annexin-
binding buffer with 5 µL FITC-annexin V and 1 µl PI working solution
(MultiSciences Biotech Co., Hangzhou, China), and then were incubated in the
dark for 15 min at room temperature. Additional 400µl binding buffer was added.
Apoptosis rate was analyzed immediately by flow cytometry (BD Biosciences,
Franklin Lakes, NJ).
4.2.7. Glucose Consumption and Lactate Release Assay
The metabolic behavior of the different cell lines was determined by
assessing extracellular amounts of glucose and lactate which were quantified
as previously described [55], using an enzymatic assay using glucose oxidase
(20U/mL) and a chromogenic solution. During glucose consumption and lactate
release assay measurements, tumor cells were firstly seeded on flat-bottom 24-
well plates (500, 000 cells/well) in normal DMEM. cells were then treated with
the DMEM medium containing compound 5o, CHC and quercetin for 4, 8, 12,
24 hours, respectively. At the end of the glucose consumption and lactate
release experiments, cell supernatants were centrifuged using deproteinizing