Coumarin carboxylic acids as monocarboxylate transporter 1 inhibitors: In vitro and in vivo studies as potential anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2016.05.054

Novel N,N-dialkyl carboxy coumarins have been synthesized as potential anticancer agents via inhibition of monocarboxylate transporter 1 (MCT1). These coumarin carboxylic acids have been evaluated for their in vitro MCT1 inhibition, MTT cancer cell viability, bidirectional Caco-2 cell permeability, and stability in human and liver microsomes. These results indicate that one of the lead candidate compounds 4a has good absorption, metabolic stability, and a low drug efflux ratio. Systemic toxicity studies with lead compound 4a in healthy mice demonstrate that this inhibitor is well tolerated based on zero animal mortality and normal body weight gains compared to the control group. In vivo tumor growth inhibition studies in mice show that the candidate compound 4a exhibits significant single agent activity in MCT1 expressing GL261-luc2 syngraft model but doesn't show significant activity in MCT4 expressing MDA-MB-231 xenograft model, indicating the selectivity of 4a for MCT1 expressing tumors.

Full text of DOI:10.1016/j.bmcl.2016.05.054

Accepted Manuscript
Coumarin carboxylic acids as monocarboxylate transporter 1 inhibitors: In vi-
tro and in vivo studies as potential anticancer agents
Shirisha Gurrapu, Sravan K. Jonnalagadda, Mohammad A. Alam, Conor T.
Ronayne, Grady L. Nelson, Lucas N. Solano, Erica A. Lueth, Lester R. Drewes,
Venkatram R. Mereddy
PII:
S0960-894X(16)30539-X
http://dx.doi.org/10.1016/j.bmcl.2016.05.054
BMCL 23913
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
3 May 2016
18 May 2016
Please cite this article as: Gurrapu, S., Jonnalagadda, S.K., Alam, M.A., Ronayne, C.T., Nelson, G.L., Solano, L.N.,
Lueth, E.A., Drewes, L.R., Mereddy, V.R., Coumarin carboxylic acids as monocarboxylate transporter 1 inhibitors:
In vitro and in vivo studies as potential anticancer agents, Bioorganic & Medicinal Chemistry Letters (2016), doi:
http://dx.doi.org/10.1016/j.bmcl.2016.05.054
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Coumarin carboxylic acids as
monocarboxylate transporter 1 inhibitors: In
vitro and in vivo studies as potential
anticancer agents
Shirisha Gurrapu, Sravan K Jonnalagadda, Mohammad A. Alam, Conor T. Ronayne, Grady L. Nelson, Lucas N.
Solano, Erica A. Lueth, Lester R. Drewes, and Venkatram R. Mereddy

Bioorganic & Medicinal Chemistry Letters
Coumarin carboxylic acids as monocarboxylate transporter 1 inhibitors: In vitro
and in vivo studies as potential anticancer agents
Shirisha Gurrapua, Sravan K. Jonnalagaddaa, Mohammad A. Alamb, Conor T. Ronayneb, Grady L. Nelsona,
Lucas N. Solanoa , Erica A. Luethb , Lester R. Drewesa,c , and Venkatram R. Mereddya,b,d 
a Integrated Biosciences Graduate Program, University of Minnesota, Duluth, MN 55812
b Department of Chemistry and Biochemistry, University of Minnesota Duluth, Duluth, MN 55812
c Department of Biomedical Sciences, Medical School Duluth, University of Minnesota Duluth, MN 55812
dDepartment of Pharmacy Practice & Pharmaceutical Sciences, University of Minnesota, Duluth, MN 55812
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Novel N,N-dialkyl carboxy coumarins have been synthesized as potential anticancer agents via
inhibition of monocarboxylate transporter 1 (MCT1). These coumarin carboxylic acids have
been evaluated for their in vitro MCT1 inhibition, MTT cancer cell viability, bidirectional Caco-
2 cell permeability, and stability in human and liver microsomes. These results indicate that one
of the lead candidate compounds 4a has good absorption, metabolic stability, and a low drug
efflux ratio. Systemic toxicity studies with lead compound 4a in healthy mice demonstrate that
this inhibitor is well tolerated based on zero animal mortality and normal body weight gains
compared to the control group. In vivo tumor growth inhibition studies in mice show that the
candidate compound 4a exhibits significant single agent activity in MCT1 expressing GL261-
luc2 syngraft model but doesn’t show significant activity in MCT4 expressing MDA-MB-231
xenograft model, indicating the selectivity of 4a for MCT1 expressing tumors.
Keywords:
N,N-dialkyl carboxy coumarin, tumor glycolysis,
monocarboxylate transporter 1, MCT, glioblastoma,
GL261-luc2, triple negative breast cancer, MDA-
MB-231
2009 Elsevier Ltd. All rights reserved.
Numerous cancer chemotherapeutics have been developed
over the past 50 years to improve the quality of patient care and
overall survival rate. However, cancer continues to be one of the
major killers throughout the world. Suboptimal efficacy,
unacceptable side effects, and development of chemo-resistance
are some of the reasons for treatment failure and patient
mortality. Hence, new candidate compounds with novel
mechanisms of action, low side effects, and that work on all
stages of tumors are urgently needed.
Recent studies indicate that alteration in cellular metabolism
is a crucial hallmark of cancer development.1,2 Therefore, tumor
metabolism is an attractive target for developing new cancer
therapeutics.3,4 The metabolic properties of cancer cells differ
significantly from those of normal cells. In normal differentiated
tissues, cellular energy is generated mainly via an efficient
oxidative phosphorylation (OxPhos). In contrast, cancer cells
pursue vigorous glycolysis even in the presence of sufficient
amounts of oxygen (Warburg effect, WE).5-10 Glycolysis
generates only two moles of ATP per one mole of glucose
compared to OxPhos, which can produce up to 38 moles of ATP.
To compensate this energy inefficiency, cancer cells upregulate
glycolytic enzymes to keep up with the energy requirements of
of glycolytic activity is essential for survival, fueling anabolic
pathways, tumor advancement, resistance to apoptosis, and
invasion and metastasis of cancer cells. 5-11
Because cancer cells are mainly dependent on glycolysis for
their survival and propagation, they need to export the glycolysis
end products pyruvic and lactic acids to avoid cytoplasmic
acidification that may lead to apoptosis. To accomplish this task,
cancer cells upregulate proton-coupled membrane proteins called
monocarboxylate transporters (MCTs), which are responsible for
transmembrane shuttling of small carboxylates such as lactate,
pyruvate, and ketone bodies.12-18 There are 14 known isoforms of
MCTs, but MCTs 1-4 are responsible for transporting these
carboxylates. They are also implicated in influx and efflux of
lactate by cancer-associated stromal fibroblasts and epithelial
cancer cells for energy generation.19-22 Elevated expression of
MCT1 has been identified in a large number of cancers and,
therefore, this transporter is a major selective target for broad-
spectrum cancer treatment.23-34 In this regard, we recently
developed a series of MCT1 inhibitors based on the α-hydroxy-4-
cyanocinnamic acid (CHC) template for potential anticancer
applications.23 Although our earlier CHC based MCT1 inhibitors
are potent, the lead compounds suffer from poor oral
bioavailability and toxicity at high concentrations. In this regard,
cell proliferation and tumor progression. Maintaining a high level
———
 Corresponding author. Tel.: +1-218-726-6776; fax: +1-218-726-7394; e-mail: vmereddy@d.umn.edu

we envisioned to utilize a similar but bicyclic coumarin template
to improve absorption, distribution, metabolism and elimination
properties, decrease systemic toxicity and improve anticancer
efficacy. Coumarin is a key structural unit with good
pharmaceutical properties that is found in many important
medicinal molecules.35-37 Structure-activity relationship studies
using our first generation CHC template indicated that placing
N,N-dialkyl/diaryl groups at the 4-position demonstrated the most
optimized structural moiety for potent MCT1 inhibition.
Therefore, we synthesized carboxy coumarins with N,N-dialkyl
substitution at the 7-position as second generation MCT1
inhibitors (Figure 1).25
We synthesized N,N-dialkyl carboxy coumarins starting from
the alkylation of o-aminophenol 1 to obtain N,N-dialkylated-o-
aminophenols 2. These alkylated aminophenols were formylated
under Vilsmeier Haack conditions with POCl3 and DMF to
obtain the corresponding aldehydes 3 in 60-70% yields. The
aldehydes 3, upon Knoevenegal condensation with diethyl
malonate, followed by the treatment with aqueous NaOH at
100oC and adjusting pH to 7 at room temperature provided
carboxy coumarins 4a-4e (Scheme 1).38
We then carried out the MCT1 inhibition study of 4a-4e using
L-[14C]-lactate uptake on rat brain endothelial-4 cells that
predominantly express MCT1.23 This transport inhibition study
revealed carboxy coumarins 4a-4e as potent inhibitors of MCT1
at nanomolar to low micromolar concentrations (Table 1).
TABLE 1: MCT1 IC50*(µM) values of amino carboxy
coumarins
Compound MCT1 IC50 in µM
0.09±0.01
0.38±0.08
0.45±0.05
0.17±0.04
0.21±0.02
4a
4b
4c
4d
4e
Figure 1: CHC and coumarin derivatives.
Here, we report on the pre-clinical evaluation of novel
coumarin carboxylic acids. Included is the synthesis, in vitro
MCT1 inhibition, cytotoxicity against two cancer cell lines, in
vitro Caco-2 permeability and metabolic stability in mouse and
human liver microsomes. Systemic toxicity studies in CD-1 mice,
in vivo anticancer efficacy in glioblastoma GL261-luc2 syngraft
and MDA-MB-231-luc xenograft mouse models are also
reported.
* Average±SEM of minimum three separate experimental values
Encouraged by excellent inhibition of MCT1, we then
evaluated cytotoxicity of compounds 4a-4e in two cancer cell
lines that are known to express either MCT1 or MCT4. For this
purpose, a predominantly MCT1 expressing cell line, GL261-
luc2, and an MCT4 expressing cell line, MDA-MB-231 were
chosen (Figure 2).39 GL261 is an important and a well-
established mouse glioblastoma (GBM) cell line that closely
mimics human gliomas.40,41 MDA-MB-231 is a triple negative
breast cancer cell line derived from human tissue.
Results and Discussion
Figure 2: Western blot analysis of MCT1 and MCT4 expression
in GL261-luc2 and MDA-MB-231 cell lines.
To determine in vitro toxicity, we evaluated cell viability
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay.42,43 This colorimetric assay measures the
reduction of MTT to formazan by cellular mitochondrial
reductase as a measure of cell viability. Compounds 4a-4e were
found to be generally non-cytotoxic at high concentrations as can
be expected from MCT inhibitors (Table 2). These results
suggest that under in vitro conditions, the cancer cells are able to
adapt their metabolism to other pathways to maintain viability.
Furthermore, it was reported that potent inhibition of MCT1 did
not have any appreciable cytotoxic properties for several solid
tumor cell lines.44 In contrast, in in vivo systems where tumor
Scheme 1: Synthesis of 7-(dialkylamino)-2-oxo-2H-chromene-3-
carboxylic acid. a) alkyl bromide, K2CO3, DMSO or EtOH, 80oC,
12h; b) POCl3, DMF, 0oC to 80oC, 2-4h; c) (i) diethyl malonate,
piperidine, CH3COOH, EtOH, 80oC, 8-12h (ii) 10% NaOH,
100oC, 2h (iii) 3M HCl, pH 7.0; reported yields are from the
reaction of 3 to 4.

hypoxia is prevalent, cancer cells are dependent on ATP
generation via vigorous glycolysis for essential metabolites,
survival, and proliferation. Hence, chronic administration of an
MCT1 inhibitor in vivo should hamper the glycolytic process,
leading to severe energy crisis and tumor growth inhibition.
Based on its excellent MCT1 inhibition, slightly enhanced
cytotoxicity, and favorable solubility, compound 4a was selected
for further in vitro and in vivo evaluation.
enzymes that are responsible for drug metabolism. 4a exhibited
good metabolic stability (half-life >60 minutes) in both human
and mouse liver microsomes (Tables 4 & 5). Propranolol,
imipramine, verapamil, and terfenadine were used as controls
with high, high, medium, and low metabolic stabilities,
respectively, in human liver microsomes.
Table 4: Intrinsic clearance in human liver microsomes (0.1µM)
TABLE 2: MTT assay IC50* values of amino carboxy coumarins
in MDA-MB-231, GL261-luc2 and MIA PaCa-2 cell lines
Intrinsic clearance (liver microsomes, human)
Half-life*
Trial 1 Trial 2
922.2
334.4
213.9
21.1
Clint
Compound
Mean
>60
>60
>60
21
Compound GL261-luc2 MDA-MB-231
>60
373
194.4
21.5
9.1
<115.5
<115.5
<115.5
324.8
4a
>0.25
>0.25
>0.25
>0.25
>0.25
0.24±0.01
>0.25
>0.25
>0.25
4a
4b
4c
4d
4e
Propranolol
Imipramine
Verapamil
Terfenadine
9.8
9
736.8
>0.25
* Half-life is reported in minutes, mean value of two experiments
*IC50 values reported in mM, average ± SEM of minimum three
separate experimental values
Table 5: Intrinsic clearance in mice liver microsomes (0.1µM)
We then determined its potential oral bioavailability using
bidirectional Caco-2 cell monolayer permeability, and its
metabolic stability in human and mouse liver microsomes
(Tables 3-5).45,46 Caco-2 cell permeability assays for 4a in both
apical to basolateral (A-B) and basolateral to apical (B-A)
directions were carried out to predict its potential oral
bioavailability. Caco-2 is a heterogeneous human epithelial
colorectal adenocarcinoma cell line that mimics the human
enterocytic intestinal layer.45 This assay estimates human
intestinal permeability and drug efflux ratio of the compounds.
4a showed relatively low A-B and moderate B-A permeability
(Table 3). Based on this study, 4a exhibits much better
absorption (~30%) compared to poor absorption of first
generation MCT1 inhibitors (3-6%, data not shown). Propranolol
(highly permeable), labetalol (moderately permeable), ranitidine
(poorly permeable), and colchicine (P-glycoprotein substrate)
were used as controls. The efflux ratio was calculated using the
formula Papp(B-A)/Papp(A-B) and efflux ratio >2 signifies that
drug efflux is occurring. 4a has an efflux ratio of 0.2 indicating
that the efflux rate is very low and 4a may not be a good
substrate for drug efflux transporters, which is beneficial for
providing anticancer efficacy.
Intrinsic clearance (liver microsomes, mouse)
Half-life*
Trial 1 Trial 2
Clint
Compound
Mean
>60
9
16
18
90.6
9.1
15.7
17.6
7.5
73.5
9.5
15.4
17.5
6.1
<115.5
744.5
446.2
394.6
1027.4
4a
Propranolol
Imipramine
Verapamil
Terfenadine
7
* Half-life is reported in minutes, mean value of two experiments
We then evaluated the systemic toxicity profile of 4a in
healthy CD-1 mice.47 Mice were treated once daily (qd) with 4a
intraperitoneally (ip, 20mg/kg) and via oral gavage (100mg/kg).
Control mice received the vehicle (10% DMSO in saline). At the
end of the study, the treated groups did not show any visible
toxic effects or mortality and had no significant difference in
body weights compared to the control group (Figure 3). This
study indicates the nontoxic nature of the candidate compound 4a
for in vivo anticancer applications.
Table 3: In vitro Caco-2 monolayer permeability (pH 6.5/7.4, at
10 µM)
A-B permeability (Caco-2, pH 6.5/7.4)
Permeability (10-6
Percent Recovery
cm/s)
Mean*
32
(%)
Mean*
34
Compound
4a
Propranolol 40.9
67
Labetalol
Ranitidine
Colchicine
8.5
ND**
0.3
66
ND**
75
B-A permeability (Caco-2, pH 6.5/7.4)
6.4
55
80
72
85
81
4a
Figure 3: Body weight changes in systemic toxicity study of
compound 4a in CD-1 mice.
Propranolol 41.5
Labetalol
Ranitidine
Colchicine
36.5
3.7
15.3
We then evaluated anticancer efficacy of 4a in a flank-based
GL261-luc2 tumor syngraft model.47-49 Group 1 was administered
with 4a (20 mg/kg, ip, qd), group 2 was treated with the
clinically used brain tumor drug temozolomide (20mg/kg, ip, qd)
and the control group was given vehicle. At the end of the study,
the tumor growth inhibition of compound 4a and temozolomide
was found to be 77% and 81%, respectively, compared to the
* mean of two experiments; **ND = not determined
We then determined the metabolic stability (half-life) of 4a
in human and mouse liver microsomes in order to assess its
hepatic clearance rate because liver microsomes contain many

control group (Figure 4). This efficacy study demonstrated the
therapeutic utility of 4a for potential anticancer applications.
effects. The candidate compound 4a was evaluated for in vivo
tumor growth inhibition in predominantly MCT1 expressing
GL261-luc2 glioma syngraft, and MCT4 expressing MDA-MB-
231 xenograft mouse models. These in vivo studies indicated that
compound 4a significantly inhibited tumor growth in GL261-
luc2 model, but did not exhibit any significant activity in MDA-
MB-231-luc model. This emphasizes the importance of high
MCT1 expression for the efficacy of these inhibitors. Owing to
the importance of glycolysis in tumor progression and the
elevated expression of MCT1 in several cancers, we believe that
these inhibitors have good potential to be developed as broad-
spectrum anticancer agents. 4a can be used as a single agent and
can also be combined with other chemotherapeutic agents with
different mechanisms of action to realize their synergistic
potential in cancer treatment.
Acknowledgments
This work was supported by University of Minnesota Duluth.
Figure 4: Tumor growth inhibition study with compound 4a in
GL261-luc2 tumor syngraft model. N = 6; *P < 0.05.
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were administered with compound 4a at two different dosages of
20 mg/kg, ip, qd and 100mg/kg, oral gavage, qd. Group 3 was
treated with the clinically used breast cancer drug doxorubicin
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(day-19), the tumor growth inhibition in groups 1, 2, and 3 were
found to be 10, 13, and 50% respectively, compared to the
control group (Figure 5). This study indicates that compound 4a
doesn’t exhibit significant tumor growth reduction in a
predominantly MCT4 expressing tumor model.
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penicillin-streptomycin (50U/ml, 50µg/ml). Cells (5 x 103 cells/well)
were seeded in 96-well plate and incubated at 37˚C and 5% CO2 for 24
hours. Test compounds were added to the wells in replicate and
incubated for 72 hours. 10µL MTT (12mM in 1X PBS) was added in
each well and incubated for 4 hours before adding 100µL SDS
(1g/10mL 0.01N HCl) to dissolve formazan precipitate and incubated
for further 4 hours. Absorbance was recorded at 570nm. Percent
survival was calculated using the formula %survival = (abs test
compound/abs of DMSO control) x 100. IC50 was obtained using
GraphPad by plotting log[concentration] on x-axis and % survival on y-
axis.
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47. Ethical Considerations: The experimental procedure involving
animals that were conducted at the University of Minnesota Duluth was
in compliance with the U.S. National Institutes of Health Guide for Care
and Use of Laboratory Animals and approved by the Institutional
Animal Care and Use Committee (IACUC). Studies with protocols
1311-31063A (systemic toxicity, Figure 3) and 1312-31108A (GL261-
luc2 syngraft, Figure 4) were conducted at University of Minnesota.
MDA-MB-231-luc xenograft study (Figure 5) was conducted by
GenScript Corporation (Piscataway, NJ) according to their approved
IACUC protocol GS-PAMD1401SN052.
33. Draoui, N.; Feron, O. Dis. Model Mech. 2011, 4, 727.
34. Hao, J.; Chen, H.; Madigan, M. C.; Cozzi, P. J.; Beretov, J.; Xiao, W.;
Delprado, W. J.; Russell, P. J.; Li, Y. Br. J. Cancer, 2010, 103, 1008.
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38. Representative procedure for the synthesis of 7-(dibenzylamino)-2-
oxo-2H-chromene-3-carboxylic acid 4a: To
a solution of 3-
48. Tumor growth inhibition studies in GL261-luc2 syngraft model:
Tumor cells suspended in 1:1 matrigel-PBS were injected onto the right
flank of C57BL/6 mice (5x106 GL261-luc2 cells). The mice were
randomly assigned into groups (n=6, male and female mice). Treatment
was started when tumor volume reached 200mm3. Tumors were
measured by caliper every two or three days and tumor volumes
calculated using the formula V = (ab2)/2 where ‘a’ is the long diameter
of the tumor and ‘b’ is the short diameter of the tumor. Mice were
euthanized at the end of the study and tumors were isolated and
weighed. The inhibition amount was determined using the formula
%inhibition = [(C-T)/C] x 100 where C is average tumor weight of the
control group and T is the average tumor weight of the test group.
49. Statistical Analysis: Statistics were computed using GraphPad Prism
version 6.0. Mann-Whitney test was used to compare the treated and
untreated groups for all in vivo tumor syngraft/xenograft studies. A P-
value of <0.05 was considered significant.
50. Tumor growth inhibition studies in MDA-MB-231 xenograft model:
Tumor cells suspended in 1:1 matrigel-PBS were injected on right flank
of female SCID mice (10x106 MDA-MB-231 cells). The mice were
randomly assigned into groups (n=6). Treatment was initiated when the
average tumor volume was ~100mm3. Mice were euthanized at the end
of the study and tumors were isolated and weighed. Tumor growth
inhibition was calculated as above in reference 49.
aminophenol (10 mmol) in 10 mL DMSO (ethanol-H2O for other alkyl
bromides), was added benzyl bromide (40 mmol), potassium carbonate
(20 mmol) and refluxed at 80 oC for 12 hours. Upon the completion of
the reaction, the reaction mixture was extracted with ethyl acetate and
water. The organic layer was dried with anhydrous Mg2SO4 and
evaporated to obtain the 3-(dibenzylamino)phenol. The product was
purified via column chromatography. To
a
solution of 3-
(dibenzylamino)phenol (10 mmol) in DMF (60 mmol) was added
phosphorous oxychloride dropwise at 0oC and the reaction mixture was
refluxed at 80oC for 2-4 hours. The reaction was quenched in a saturated
solution of sodium carbonate and the solid was filtered and washed with
hexanes to obtain 4-(dibenzylamino)-2-hydroxybenzaldehyde. To a
solution of this aldehyde (10 mmol) in 20 ml ethanol, was added diethyl
malonate (20 mmol), acetic acid (5 drops) and piperidine (13 mmol) and
refluxed for 8-12 h at 80 °C. Upon the completion of the reaction, the
above solution was evaporated and extracted with ethyl acetate. The
organic layers were dried with anhydrous MgSO4 and evaporated. The
product obtained was further refluxed in 20 ml of 10% NaOH solution.
The reaction was quenched with 3M HCl (pH 7.0) and extracted with
ethyl acetate. The compound was purified by column chromatography
(eluted with 100% ethyl acetate) and recrystallized. 1H NMR (500 MHz,
CDCl3): δ 12.31 (s, 1H), 8.69 (s, 1H), 7.48-7.23 (m, 11H), 6.86 (dd, J =
8.5, 1.5 Hz, 1H), 6.67 (s, 1H), 4.84 (s, 4H); 13C NMR (125 MHz,
CDCl3): 165.53, 164.24, 157.89, 155.60, 150.75, 135.84, 132.23,
129.46, 128.16, 126.52, 112.02, 109.79, 107.24, 98.63, 55.03; Anal.
Calcd for C24H19NO4.HCl (421.88): C 68.33, H 4.78, N 3.32 Found: C
69.22, H 4.88, N 3.43.
39. Western blot analysis of MCT1 and MCT4 expression: Cultured
cells were scraped from culture flasks and immediately frozen. Cells
were thawed on ice and solubilized in 200μL SDS boiling buffer, then
centrifuged for 2 min at high speed. The supernatant was collected and
diluted 1:5 with deionized H2O and assayed for protein using the BCA
protocol. A volume containing 10μg was loaded on SDS PAGE gels
(Novex) for standard electrophoresis (40 min at 200 V). Proteins were
transferred from the gel to nitrocellulose membrane under denaturing
conditions (200 mA for 1.75 hr). MCT1 and MCT4 were detected using
specific antibodies (rabbit polyclonal IgG antibody for MCT1, sc-
50324; rabbit polyclonal IgG antibody for MCT4, sc50329; Santa Cruz,