Monocarboxylate transporter 1 inhibitors as potential anticancer agents

Article abstract of DOI:10.1021/acsmedchemlett.5b00049

Potent monocarboxylate transporter 1 inhibitors (MCT1) have been developed based on α-cyano-4-hydroxycinnamic acid template. Structure-activity relationship studies demonstrate that the introduction of p-N, N-dialkyl/diaryl, and o-methoxy groups into cyanocinnamic acid has maximal MCT1 inhibitory activity. Systemic toxicity studies in healthy ICR mice with few potent MCT1 inhibitors indicate normal body weight gains in treated animals. In vivo tumor growth inhibition studies in colorectal adenocarcinoma (WiDr cell line) in nude mice xenograft models establish that compound 27 exhibits single agent activity in inhibiting the tumor growth.

Full text of DOI:10.1021/acsmedchemlett.5b00049

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Letter
Monocarboxylate Transporter 1 Inhibitors as Potential Anti-cancer Agents
Shirisha Gurrapu, Sravan K Jonnalagadda, Mohammad A Alam, Grady L
Nelson, Mary G Sneve, Lester R Drewes, and Venkatram Reddy Mereddy
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00049 • Publication Date (Web): 19 Mar 2015
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Monocarboxylate Transporter 1 Inhibitors as Potential Anti-
cancer Agents
Shirisha Gurrapu,a Sravan K. Jonnalagadda,a Mohammad A. Alam,b Grady L. Nelson,a Mary G.
Sneve,c Lester R. Drewes,*c and Venkatram R. Mereddy*a, b, d
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aIntegrated Biosciences Graduate Program, University of Minnesota, Duluth, MN 55812
bDepartment of Chemistry and Biochemistry, University of Minnesota Duluth, Duluth, MN 55812
cDepartment of Biomedical Sciences, Medical School Duluth, University of Minnesota, MN 55812
dDepartment of Pharmacy Practice & Pharmaceutical Sciences, University of Minnesota, Duluth, MN 55812
KEYWORDS Warburg effect, reverse Warburg effect, monocarboxylate transporter - 1, α-cyano-4-hydroxy cinnamic
acid, colorectal adenocarcinoma.
ABSTRACT Potent monocarboxylate transporter 1 inhibitors (MCT1) have been developed based on α-cyano-4-hy-
droxycinnamic acid template. Structure activity relationship studies demonstrate that the introduction of p-N, N-dialkyl/
diaryl and o-methoxy groups into cyanocinnamic acid has maximal MCT1 inhibitory activity. Systemic toxicity studies in
healthy ICR mice with few potent MCT1 inhibitors indicate normal body weight gains in treated animals. In vivo tumor
growth inhibition studies in colorectal adenocarcinoma (WiDr cell line) in nude mice xenograft models establish that com-
pound 27 exhibits single agent activity in inhibiting the tumor growth.
Vigorous glycolysis is the hallmark of many advanced
stage tumors.1-5 Malignant tumors are heterogeneous in na-
ture and tumor microenvironment contains aerobic and
hypoxic regions.1 Tumor hypoxia leads to cancer advance-
ment, metastasis, treatment failure, and patient mortality
as these cells become resistant to standard chemo- and ra-
diation therapy. Under hypoxic conditions, cancer cells
consume glucose that is metabolized to lactate, ketone and
other energy rich molecules.1 The transport of these energy
rich metabolites occurs via monocarboxylate transporters
(MCTs).1,4,6,7 They are members of the solute carrier 16 gene
family consisting of 14 known MCT isoforms (SLC16A1-
14).6,7 Of these, MCTs 1-4 have been shown to transport
monocarboxylates such as lactate, pyruvate, and some ke-
tone bodies.1,6,7 MCT1 expression is elevated in an array of
human tumors and therefore, these transporters can be
major selective targets for anti-tumor therapy.1,8-14 Re-
cently, Lisanti et. al. have carried out extensive studies to
explain the glycolytic process in epithelial cancer cells.15-18
These studies strongly indicate that cancer cells initiate ox-
idative stress in their neighboring fibroblasts resulting in
mitochondrial dysfunction and aerobic glycolysis (War-
burg Effect). These fibroblasts then produce lactate and py-
ruvate that are taken up by adjacent cancer cells via MCT1
for mitochondrial oxidative phosphorylation and further
proliferation. In essence, tumor associated stromal fibro-
blasts and epithelial cancer cells develop a metabolic sym-
biotic relationship (Reverse Warburg Effect).15-18
Owing to the importance of MCT1 in the above men-
tioned tumor related biological events, development of
MCT1 inhibitors should be of immense importance in
broad-spectrum cancer treatment. In this regard, we initi-
ated a project on the discovery of novel MCT1 inhibitors as
potential anti-cancer agents based on α-cyano-4-hy-
droxycinnamic acid (CHC).19 Although CHC has been in bi-
ochemical usage as a low affinity MCT1 inhibitor (IC50 >100
µM), the derivatives of CHC have not been studied to im-
prove its potency. We envisioned the systematic design,
synthesis and evaluation of several CHC analogs to under-
stand the structure-activity relationship (SAR) and obtain
lead molecules that inhibit MCT 1 at low concentrations for
potential development as anti-cancer agents.
CHC-Based Analogs: There are five positions of substi-
tution on the CHC template (Scheme 1A) and we chose p-
hydroxyl replacement as the starting point. For initial stud-
ies, hydroxyl group was substituted with alkyl (methyl,
ethyl, 1-2), electron withdrawing (F, Cl, Br, CN & NO2, 3-7)
and electron donating groups (methoxy, 3, 4-methylenedi-
oxy, 8-9). These CHC derivatives of α-cyanocinnamic acids
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were synthesized from corresponding aldehydes via
72% yield. To our delight, 10 exhibited potent MCT1 inhi-
Knoevenagel type condensation (1-9, Scheme 1B).20
bition activity with a mean IC50= 0.14 µM. Taking 10 as a
lead derivative, we further explored the SAR (Table 1) by
structural modification on the nitrogen atom. In this re-
gard, we synthesized several p- N, N-dialkyl and aryl sub-
stituted cyanocinnamic acids starting from aniline
(Scheme 1C). Di-alkylation of aniline followed by Vils-
meier–Haack formylation23 and subsequent base mediated
condensation with cyanoacetic acid provided cinnamic ac-
ids 11-21 in good yields (Scheme 1C & Table 1). Com-
pounds 11-21 upon evaluation of their MCT1 inhibition ac-
tivities on RBE4 cell line exhibited a clear pattern of SAR.
From these studies, it was found that increasing the alkyl
chain increased the potency up to four carbon chain length
(11-13, 13-66 nM, Table 1). Further increase in the chain
length to pentyl 15 and hexyl 16 decreased the activity. Aryl
substituted cyanocinnamic acid 19 also showed excellent
activity (26 nM). Cyclic amines such as pyrrolidine 20 and
piperidine 21 decreased the activity to a moderate range of
IC50 values (70 and 142 nM, Table 1).
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2
3
4
5
6
7
8
The compounds 1-9 were evaluated for their MCT1 inhi-
bition activity via [14C]-lactate uptake assay on rat brain en-
dothelial 4 cells (RBE4). We utilized this cell line for initial
lactic acid transport studies as they highly express MCT1 as
confirmed by Western Blotting.21,22 Gratifyingly, com-
pounds 1-9 have exhibited several times higher potency
(~0.5-100 µM) than the parent CHC molecule (Scheme 1B).
9
We then explored the role of cyano (-CN), carboxylic
acid (-COOH) and olefinic functionalities in CHC by re-
placing -CN group with H atom, replacing –COOH and in-
troducing two -CN groups, removal of the -CN group, and
also reduction of the olefin. In all these cases, we did not
observe any appreciable MCT1 inhibition activity when
compared to CHC (data not shown).
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Scheme-1
Table 1. MCT1 IC50 values of N, N- dialkyl/aryl cyano-
cinnamic acids
Sl.
No
10
11
12
13
14
15
16
17
MCT1 IC50
(µM)**
X
% Yield*
N, N-dimethyl
N, N-diethyl
N, N-dipropyl
N, N-dibutyl
N, N-diisobutyl
N, N-dipentyl
N, N-dihexyl
N, N-diallyl
72
75
67
65
61
70
68
60
62
76
62
67
0.14±0.05
0.07±0.02
0.01±0.01
0.03±0.00
0.10±0.05
0.14±0.02
0.88±0.18
0.11±0.03
0.40±0.02
0.03±0.00
0.07±0.01
0.14±0.03
18
19
20
21
N, N-dipropargyl
N, N-diphenyl
Pyrrolidinyl
Piperidinyl
* Starting from aldehydes
** Average IC50 values of minimum three separate experi-
ments. Values reported as avg ± SEM
Reagents and conditions: (a) Alkylbromide, K2CO3, tetrabu-
tylammonium bromide, H2O/ethanol or isopropanol, 100oC,
8-24hrs, (b) i) POCl3/DMF 0oC-80oC, 2-4h, ii) Na2CO3, (c)
CNCH2COOH, piperidine, CH3CN, 80oC, 8 - 20 h, (d) Na-
HCO3, MeOH-H2O, rt, overnight.
Based on the significantly improved activity of N, N-di-
alkyl series, we further carried out SAR studies by intro-
ducing substitutions at 2-position of aromatic ring
(Scheme 1A). For initial studies, we started with cyanocin-
namic acid 22 derived from o-methoxy-N, N-dipropyl ben-
zaldehyde. 22 exhibited higher MCT1 inhibitory activity
(IC50= 12 nM) compared to its non-methoxy derivative 12.
To understand further SAR of this lead candidate, we in-
troduced several alkyl and aryl substitutions on the nitro-
gen atom. The required aldehydes were synthesized from
m-anisidine (Scheme 1D). Alkylation of m-anisidine with
We then introduced N, N- dialkyl group in place of hy-
droxyl moiety owing to the biological significance of nitro-
gen atom in many pharmaceutical compounds. Condensa-
tion of commercially available p-(N, N-dimethyl) benzal-
dehyde with cyanoacetic acid in the presence of piperidine
provided the corresponding cyanocinnamic acid 10 in ~
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alkyl halides in the presence of K2CO3 and catalytic tet- and weight gains were similar to the control group
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8
rabutylammonium bromide followed by Vilsmeier-Haack
formylation23 provided the corresponding aldehydes.
These aldehydes were then condensed with cyanoacetic
acid in the presence of piperidine to synthesize cyanocin-
namic acids 22–30 (Scheme 1D, Table 2). MCT1 based
[14C]-lactate uptake studies on RBE4 cells revealed that
these derivatives 22-30 have superior inhibitory activity
(IC50= 8-48 nM) when compared to their non-methoxy
homologues 10-21 (Table 1). In fact, cyanocinnamic acids
22-24 & 26 have exhibited inhibitory activity in low nM
range (8-12 nM, Table 2).
(Supporting Information). Due to the higher water solubil-
ity of sodium salts of 29 and 30, toxicity studies were car-
ried out at the higher dosage of 70 mg/kg/ip/bid. In these
cases also, no animal mortality was observed and the body
weight gains were similar to the control group (Supporting
Information).
We also carried out a preliminary pharmacokinetic anal-
ysis of 27 in ICR mice. 60 ICR mice were divided into two
groups and group-1 was administered with 27 intraperito-
neally and group-2 intragastrically at 100 mg/kg. Blood
samples were collected at different time points (n=3 for
each time point). The peak plasma concentrations were
observed at 0.25h for group-1 and 0.30h for group-2. In
both the cases, compound 27 was completely eliminated
from the system within 24h (Supporting Information).
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10
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19
20
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22
23
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26
27
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Table 2. MCT1 IC50 values of o-methoxy p-N, N- dial-
kyl/aryl cyanocinnamic acids
Encouraged by generally non-toxic nature of potent
MCT1 inhibitors in healthy animals, we next carried out the
in vivo tumor growth inhibition study with 27 using flank-
based tumor xenografts in nude mice. For initial studies,
we utilized a MCT1 expressing and highly tumorigenic col-
orectal adenocarcinoma cell line WiDr.1 These cancer cells
(7 x 106) in 1:1 PBS-matrigel were inoculated into the flank
of female BALB/c nude mice and allowed to grow until the
tumor volume reached ~100 mm3 (~3weeks). Mice were ad-
ministered with 27 by intraperitoneal injection (10 mg/kg,
bid) and by oral gavage (50 mg/kg, bid). The treatment was
continued for three weeks and tumor volumes were deter-
mined with calipers every three days (Figure 1). At the end
of the study, mice were euthanized, tumors were isolated
and weighed to validate the caliper measurements. Based
on these studies, ~29% & 45% tumor growth inhibition was
observed in ip and oral gavage groups respectively.
Sl.
No.
%
yield*
MCT1 IC50
nM**
12±1
X
Y
22
23
24
25
26
27
28
29
30
N, N-dipropyl
N, N-dibutyl
N, N-diisobutyl
N, N-dipentyl
N, N-diphenyl
N, N-diphenyl
N, N-diallyl
H
H
H
H
H
62
60
58
74
66
9±1
11±4
34±7
8±1
Na 78#
11±3
H
H
H
70
68
85
29±2
48±20
25±5
Pyrrolidinyl
Piperidinyl
* Starting from aldehydes
# Starting from acid
** Average IC50 values of minimum three separate experi-
ments. Values reported as avg ± SEM
C o m p ou n d 2 7 , 50 m g/kg,
1000
o ral ga va ge, bid
C o m p o u n d 2 7 , 1 0 m g /kg , i.p , b id
C on tro l gro up
800
600
400
200
0
We chose 26 as a representative example for further eval-
uation based on its potent MCT1 inhibitory profile. To im-
prove the water solubility, we prepared its sodium salt 27
by treating the corresponding acid 26 with NaHCO3 in
methanol-water. The sodium salt 27 has water solubility of
~1 mg/mL. Compound 27 retained its potent MCT1 inhibi-
tory activity (11 nM, Table 2). Similarly, we have also syn-
thesized sodium salts of 29 and 30 and they exhibited water
solubility of >7 mg/ml. Having achieved the initial goal of
discovering potent MCT1 inhibitors, we then carried out
systemic toxicity analysis of some of these derivatives in
healthy ICR mice.
*
*
*
*
*
0
2
5
9
12
16
19
22
D a y s a fte r tre a tm e n t
Figure 1: In vivo efficacy with 27 as a single agent in WiDr tu-
mor xenograft model: WiDr cells were implanted subcutane-
ously in the right flanks of female BALB/c nude mice. After
tumors reached ~100 mm3, mice were randomly assigned to
three groups (N=8 mice per group). Group 1 was administered
with 50 mg/kg, oral gavage, bid, group 2 was treated with 10
mg/kg, ip, bid. Tumor volumes were recorded every 3-4 days.
The study was carried out for 22 days. Mann-Whitney test was
used to compute significance. * P ≤ 0.05
Compound 27 was chosen for initial studies and the mice
were treated with 27 in 0.1% DMSO in saline for three
weeks (6.67 mg/kg, ip, bid). All mice survived and weight
gains were similar when compared to the control group
(Supporting Information). We then carried out another
study with increased dosages of 50 mg/kg, oral gavage, bid,
and 50 mg/kg, ip, qd. In these cases also, all mice survived
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agents with higher affinity and specificity than previously
available.
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2
3
4
5
6
7
8
Similarly, another in vivo tumor growth inhibition study
was conducted by starting the treatment from day-6 fol-
lowing WiDr tumor inoculation. This time, 27 was admin-
istered at a higher dosage of 100 mg/kg, oral gavage, qid
and 50 mg/kg, ip, bid in groups 1 & 2 respectively. By the
end of the study (21 days, tumor volumes Figure 2), 56%
and 45% tumor growth inhibition was observed in groups
1 & 2 respectively based on the weights of the isolated tu-
mors.
ASSOCIATED CONTENT
Supporting Information. Synthetic methods and proce-
dures of all the compounds, spectroscopic data and elemental
analysis, in vitro and in vivo biological protocols. This material
is available free of charge via the Internet at
http://pubs.acs.org.
9
AUTHOR INFORMATION
Corresponding Author
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8 0 0
C o m po u n d 27 , 10 0 m g /kg , oral g a va g e , q id
* (V.R.M.) E-mail: vmereddy@d.umn.edu Tel: 218-726-6766.
*(L.R.D.) E-mail: ldrewes@d.umn.edu, Tel: 218-726-7925.
C o m p o u n d 2 7 , 5 0 m g /kg , ip , b id
C on tro l gro up
6 0 0
Funding Sources
This work was supported by Engebretson Drug Discovery Pro-
gram, College of Pharmacy, University of Minnesota; White-
side Clinical Research Institute, Duluth, Minnesota and Min-
nesota Partnership for Biotechnology & Medical Genomics.
4 0 0
*
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2 0 0
*
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*
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*
0
Notes
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1 2
1 5
1 8
2 1
The authors declare the following competing financial inter-
est: A patent application has been filed, but it has not been
licensed nor has generated any revenue to date.
D a y s a fte r tre a tm e n t
Figure 2: In vivo efficacy with 27 in WiDr tumor xenograft
model: WiDr cells were implanted subcutaneously in the right
flank of female BALB/c nude mice. Treatments were started
on day-6 of implant. Mice were randomly assigned to three
groups (N=8 mice per group). Group 1 was administered with
100 mg/kg, oral gavage, qid, group 2 received 50 mg/kg, ip, bid.
Tumor volumes were recorded every two days. The study was
carried out for 21 days. Mann-Whitney test was used to com-
pare the treated and untreated groups. * P ≤ 0.05
ABBREVIATIONS
CHC, α-cyano-4-hydroxycinnamic acid; MCT, monocarbox-
ylate transporter; IC50, the concentration of test compound
causing 50% transport inhibition; SAR, structure-activity rela-
tionship; RBE4, rat brain endothelial 4 cells; K2CO3, potassium
carbonate; NaHCO3, sodium bicarbonate; SEM, standard error
of the mean; ICR mice, mice initiated at Institute for Cancer
Research; DMSO, dimethyl sulfoxide; PBS, phosphate buff-
ered saline; qd, once daily; bid, twice daily; qid, four times
daily.
These studies clearly demonstrate that MCT1 inhibitors
have potential to develop them as anticancer agents. How-
ever, detailed mechanistic studies need to be conducted
with low MCT1 expressing cell line or with MCT1 knock out
cell lines to evaluate the in vivo anticancer efficacy of 27.
Also, studies to determine whether efficacy is directly due
to MCT1 inhibition and whether any potential off-target ef-
fects are present must be conducted.
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Monocarboxylate Transporter 1 Inhibitors as Potential Anti-
cancer Agents
Shirisha Gurrapu,a Sravan K. Jonnalagadda,a Mohammad A. Alam,b Grady L. Nelson,a Mary G.
Sneve,c Lester R. Drewes,*c and Venkatram R. Mereddy*a, b, d
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