Development of methotrexate proline prodrug to overcome resistance by MDA-MB-231 cells

Article abstract of DOI:10.1016/j.bmcl.2010.07.024

The resistance to methotrexate by a number of cancer cells such as breast cancer cell-line MDA-MB-231 due to poor permeability renders it less effective as an anticancer agent for these cells. Proline prodrug of methotrexate (Pro-MTX) was designed as a substrate of prolidase which is specific for imido bond of dipeptide containing proline and expected to penetrate MDA-MB-231 cells more efficiently. The prodrug was synthesized by solid-phase peptide synthesis method and examined as a substrate of pure prolidase as well as cell homogenate. The cytotoxicity against MDA-MB-231 and non-methotrexate resistant breast cancer cell line, MCF-7 was also examined by XTT assay. The results showed that Pro-MTX was a substrate of prolidase. It was also shown that the prodrug could be converted to parent drug methotrexate in Caco-2 and HeLa cell homogenate. When tested with Caco-2 and MCF-7 cells, Pro-MTX showed weaker cytotoxicity compared with methotrexate. But for methotrexate resistant MDA-MB-231 cells, Pro-MTX showed stronger activity than methotrexate. The results indicated that the proline prodrug of methotrexate may overcome the resistance of human breast cancer cells in culture.

Full text of DOI:10.1016/j.bmcl.2010.07.024

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5108–5112
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of methotrexate proline prodrug to overcome resistance
by MDA-MB-231 cells
*
Zhiqian Wu , Anandkumar Shah, Namrata Patel, Xudong Yuan
Division of Pharmaceutical Sciences, Arnold & Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY 11201, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The resistance to methotrexate by a number of cancer cells such as breast cancer cell-line MDA-MB-231
due to poor permeability renders it less effective as an anticancer agent for these cells. Proline prodrug of
methotrexate (Pro-MTX) was designed as a substrate of prolidase which is specific for imido bond of
dipeptide containing proline and expected to penetrate MDA-MB-231 cells more efficiently. The prodrug
was synthesized by solid-phase peptide synthesis method and examined as a substrate of pure prolidase
as well as cell homogenate. The cytotoxicity against MDA-MB-231 and non-methotrexate resistant breast
cancer cell line, MCF-7 was also examined by XTT assay. The results showed that Pro-MTX was a sub-
strate of prolidase. It was also shown that the prodrug could be converted to parent drug methotrexate
in Caco-2 and HeLa cell homogenate. When tested with Caco-2 and MCF-7 cells, Pro-MTX showed weaker
cytotoxicity compared with methotrexate. But for methotrexate resistant MDA-MB-231 cells, Pro-MTX
showed stronger activity than methotrexate. The results indicated that the proline prodrug of methotrex-
ate may overcome the resistance of human breast cancer cells in culture.
Received 7 June 2010
Revised 6 July 2010
Accepted 7 July 2010
Available online 11 July 2010
Keywords:
Proline methotrexate prodrug
Prolidase
Methotrexate resistance
Enzyme-targeted delivery
Enzyme-dependent activation
MDA-MB-231
Ó 2010 Elsevier Ltd. All rights reserved.
For chemotherapeutic drugs, poor selectivity between neoplas-
tic cells and normal cells, often leading to adverse toxicity and nar-
rower therapeutic window, has been a huge problem for many
years. In addition, drug resistance occurs due to multiple mecha-
nisms ranging from diminished cellular uptake to increased efflux
to inactivation of active agents in cancerous cells.1–3 Prodrugs can
be designed to target cancer cells over-expressing enzymes which
are able to release the parent drugs to achieve enhanced therapeu-
tic selectivity and efficacy.4,5 Drugs with poor permeability can be
transformed to highly permeable drugs by making them into pro-
drugs that are substrates of transporters to improve cellular up-
take. Many studies have proved the feasibility and efficiency of
this approach.6,7
Methotrexate (MTX) is an antitumor agent being used exten-
sively in cancer chemotherapy since 1948. The mechanism of action
is well understood at molecular level. The drug is transported into
cells by the reduced folate transporter and polyglutamylated to pre-
vent efflux, and exerts its cytotoxic effect by inhibiting dihydrofolate
reductase (DHFR). Adverse effects associated with methotrexate in-
clude severe damage to normal cells and organs, narrow therapeutic
index, poor selectivity for neoplastic cells, multidrug resistance due
to down-regulation of methotrexate polyglutamation, and de-
creased reduced folate carrier (RFC) mediated uptake.8–10 Metho-
trexate resistance by certain cancer cells significantly restricts the
effectiveness of the drug, and this phenomenon has drawn attention
from many researchers. Breast cancer cell-line MDA-MB-231 is one
of the cell lines resistant to methotrexate due to low expression level
of reduced folate carrier (RFC) which can uptake methotrexate into
the cells. Methotrexate coupled with cell penetrating peptide has
been developed to overcome MTX resistance in MDA-MB-231
cells.10 It is also found that proline prodrug of melphalan can be
better transported into MDA-MB-231 cells compared with the par-
ent drug-melphalan. Melphalan is released from its prodrug, Mel-
phalan-proline, in MDA-MB-231 cells by
a specific cytosolic
imidodipeptidase, prolidase [E.C.3.4.13.9] and responsible for the
cytotoxicity against MDA-MB-231.11,12 Although prolidase needs a
dipeptide substrate with small molecular weight, it is found that
prolidasemayhavebroader substratespecificitythanthoughtprevi-
ously.13 For example, N-(anthraquinone-2-carbonyl)-
L
-proline, a
relatively larger molecule, could evoke susceptibility to the action
of prolidase comparable to glycyl- -proline (GLY-PRO, standard
L
substrate for prolidase).13 It is possible that the proline prodrug of
methotrexate could also be transported into MDA-MB-231 and re-
leased by the similar mechanisms and overcome the methotrexate
resistance due to the deficiency of RFC. The proline prodrug of meth-
otrexate, Proline–Methotrexate (Pro–MTX), is synthesized by solid-
phase chemistry with high purity.
The solid-phase synthesis of the L-proline prodrug of metho-
trexate, Pro-MTX (1) was carried out in a stepwise fashion on
Wang resin as described in Scheme 1.14 Purity of the prodrug
was determined by HPLC. Electrospray ionization mass spectra
(ESI-MS) were obtained on a Thermoquest LCQ electrospray ioniza-
tion mass spectrometer. The observed molecular weight of the
* Corresponding author. Tel.: +1 718 780 4547; fax: +1 718 780 4586.
E-mail address: james.wu@liu.edu (Z. Wu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.024

Z. Wu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5108–5112
5109
Fmoc
H
O
O
O
N
N
Piperidine
DMF
O
Fmoc-Glu(tBu)-OH
DMF
HOBT
HBTU
TEA
Wang resin Proline-Fmoc
Fmoc
NH2
HN
O
OtBu
O
OtBu
Piperidine
DMF
O
O
N
O
N
O
O
O
Wang resin
Proline-Glu(tBu)-Fmoc
N
N
N
NH2
CH3
N
HOBT
HBTU
TEA
N
HO
NH2
O
N
N
NH2
N
N
NH2
CH3
N
CH3
N
N
N
N
N
O
NH2
O
NH2
TFA , TIS
HN
HN
O
OtBu
O
OH
Cleavage/deprotection
O
O
N
O
N
O
O
HO
(1)
Proline-Methotrxate
Wang Resin
Scheme 1. Synthesis of Pro-MTX (1).
prodrug was found to be identical to that required by the structure.
Structural identity was also confirmed using proton nuclear mag-
netic resonance spectra (1H NMR). 1H NMR spectra were obtained
with a 400 MHz Bruker NMR spectrometer.15
Methotrexate and prodrug were analyzed by reversed phase
chromatography performed at room temperature. Detection wave-
length was 274 nm. Samples were injected onto a C18 column
of reagents (A and B) were prepared. Reagent A has 50 mM Tris–
HCl buffer, 200 mM manganese chloride, 30 mM glutathione, and
porcine kidney prolidase solution. Reagent A was incubated for
30 min at 40 °C to activate the enzyme. Reagent B was prepared
by mixing 1 mM Pro-MTX and 200 mM manganese chloride. After
activation, reagent A was added to reagent B at 40 °C. Reagent A
without prolidase was mixed with reagent B to serve as control.
Cbz-Pro was also added in Reagent B at the same concentration
of Pro-MTX as inhibitor. Samples were withdrawn after 15 min,
30 min, 1 h and added into same volume of 10% TFA to quench
the reaction. Then the samples were analyzed by colorimetric
method.
(Xorbax, Eclipse XDB-C18, 4.6 Â 75 mm, 3.5
lm, Agilent, CA, Part
No. WAT066224) equipped with a analytical guard cartridge sys-
tem (Phenomenex, cartridge 4.0 Â 3.0, CA) and eluted at a flow rate
of 0.6 ml/min. Mobile phase consisted of 0.1% TFA in water/meth-
anol:water (50:50) (85/15). The HPLC equipment consisted of a
Waters 717 plus autosampler, Waters 2695 separation module,
Waters 2998 (Photodiode Array Detector), and Empower 2 soft-
ware (Water Corporation) data handling system.
The hydrolysis of Pro-MTX to methotrexate was also deter-
mined in Caco-2 and HeLa cell lines to check the prolidase activity
in these cells. The cells were grown as described in cell culture
preparation. Cell homogenate were prepared when the cells were
The commercially available prolidase from porcine kidney is
similar to human prolidase (97% identity) so it is used in hydrolysis
studies to observe the conversion from prodrug to parent drug.
Prolidase solution was prepared by suspending the lyophilized so-
lid (0.8 mg solid, 0.551 mg protein) in 50 mM cold Tris–HCl buffer
(pH 8.0 at 40 °C) to yield a 5 mg/ml solution. The assays and en-
zyme activation were carried out according to protocol provided
by the manufacturer (Sigma/Aldrich, St. Louis, MO). First, two sets
90% confluent.16 Hydrolysis studies were conducted with 500
ll
supernatant at 37 °C. To activate the prolidase, 500 ll of Reagent
A with 50 mM Tris–HCl buffer, 200 mM manganese chloride,
30 mM glutathione were added and incubated for 2 h at 37 °C.
1 mM of Pro-MTX was added and 200 ll of samples were with-
drawn after 15, 30, 60 min and added into same volume of 10%
TFA to quench the reaction. The samples were then centrifuged

5110
Z. Wu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5108–5112
at 5000 rpm for 5 min at 4 °C. Two hundred microliters of the
supernatant were analyzed by colorimetric method. In competitive
inhibition study, Cbz-Pro with the same concentration as Pro-MTX
was added as inhibitor.
By measuring the amount of proline released from the hydroly-
sis of Pro-MTX, we can determine the extent of the hydrolysis and
calculate the hydrolysis rate. Chinard’s reagent (25 g of ninhydrin
in 600 ml of glacial acetic acid and 400 ml of 6 M o-phosphoric
acid) was used according to Myara et al.17 200
was added into 200
acetic acid. The mixture was incubated at 90 °C for 10 min and
200 l of the mixture was then withdrawn for UV absorbance mea-
ll of the test sample
l
l of Chinard’s reagent and 200 l of glacial
l
l
surement at 495 nm.
The specific activity of Pro-MTX for porcine kidney prolidase
calculated from assays of released proline was 19.03 1.31 pmol/
min/
l
g. In the presence of the prolidase inhibitor Cbz-Pro, the spe-
Figure 1. Cytotoxic study of methotrexate and Pro-MTX in Caco-2 cells viability of
Caco-2 cells treated for 24 h with different concentrations of methotrexate and Pro-
MTX (mean SD, n = 3).
cific activity decreased to 8.15 1.10 pmol/min/
l
g with inhibition
of 57%. The specific activity of Pro-MTX in various cancer cell
homogenate was calculated from assays of released proline.
Caco-2 and HeLa cells exhibited similar prolidase activity (0.42
and 0.35 pmol/min/lg protein, respectively). The hydrolysis of
Pro-MTX in the presence of Cbz-Pro was significantly inhibited in
all cancer cell homogenates examined. In Caco-2 cells, the specific
activity was reduced to 0.28 pmol/min/
of 33%. In HeLa cells, the specific activity was reduced to
0.19 pmol/min/ g protein with inhibition of 46% (Table 1).
lg protein with inhibition
l
Methotrexate prodrug, Pro-MTX was examined for their cyto-
toxicity against Caco-2, MCF-7, or MDA-MB-231 cells to compare
with the parent drug, methotrexate.18 Cell viability was plotted
as a function of drug concentration in the following figures. When
tested with Caco-2 cells, Pro-MTX shows weaker activity compared
with methotrexate. Pro-MTX has estimated EC50 value of 5.6
lM
while methotrexate has estimated EC50 value of 1.1 M. This is also
l
confirmed by other studies showing methotrexate amino acid pro-
drugs are generally weaker than the parent drug for their anti-pro-
liferation activity. The results for MCF-7 cells are similar with
Figure 2. Cytotoxic study of methotrexate and Pro-MTX in MCF-7 cells viability of
MCF-7 cells treated for 24 h with different concentrations of methotrexate and Pro-
MTX (mean SD, n = 3).
Caco-2 cells. Methotrexate has estimated EC50 value of 0.9
lM
while Pro-MTX shows estimated EC50 value of 5.2 M. But for
l
methotrexate resistant MDA-MB-231 cells, Pro-MTX shows stron-
ger activity than methotrexate. Pro-MTX has estimated EC50 value
of 11
lM while methotrexate shows estimated EC50 value of
80 M. The results indicate that the proline prodrug of methotrex-
l
ate may overcome the resistance of human breast cancer cells in
culture (Figs. 1–3).
Prodrug strategy has been used to target specific enzymes that
are highly expressed in certain tissues to reduce toxicity and in-
crease efficacy of the drug. Prolidase is one of the enzymes being
studied extensively. It was found by Amidon et al. that prolidase
activities with GLY-PRO as substrate in different cancerous cells
correlate very well with the expression levels of prolidase in these
cells.5 Proline prodrugs of melphalan and others with or without
linker were synthesized and studied by Bielawska11–13,19–21 and
Amidon5,22,23 et al. The prodrugs were found to be good substrate
of prolidase with significant cytotoxicity against MCF-7, Mela-
noma, and MDA-MB-231 cell lines. It is found that the proline
prodrug of melphalan was more effectively transported into the
Figure 3. Cytotoxic study of methotrexate and Pro-MTX in MDA-MB-231 cells
viability of MDA-MB-231 cells treated for 24 h with different concentrations of
methotrexate and Pro-MTX (mean SD, n = 3)..
Table 1
Specific activity of Pro-MTX in the presence and absence of Cbz-Pro in prolidase and
various cancer cell lines (expressed as pmol/min/lg of protein, mean SD, n = 3)
Cell lines
Specific activity
with out Cbz-Pro
Specific activity
with Cbz-Pro
% Inhibition
MDA-MB-231 cells with higher cytotoxicity compared to melpha-
lan.12 The nature of the transport is unknown currently, but proline
should play an important role in improving transport. Other pro-
line prodrugs, such as proline-linked nitrosoureas, also exhibit
Prolidase
Caco-2
HeLa
19.03 1.31
0.42 0.11
0.35 0.08
8.15 1.10
0.28 0.08
0.19 0.07
57
33
46

Z. Wu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5108–5112
5111
stronger cytotoxicity against MDA-MB-231 cell line compared to
MCF-7. Whether the proline improved cellular transport is not con-
firmed, but it is found that the prolidase activity in MDA-MB-231
cell line was threefold higher than in MCF-7 cell.20 MDA-MB-231
was obtained from a patient who developed resistance while being
treated with methotrexate. It is an ideal cell line to study metho-
trexate resistant cancer.24 The mechanism of the resistance was
found to be lack of reduced folate carrier (RFC) expressed on
MDA-MB-231 and that makes it more attractive to overcome the
resistance by a prodrug utilizing other transport route.
permeability. The results indicated that the proline prodrug of
methotrexate may overcome the resistance of human breast can-
cer cells in culture.
References and notes
1. Stavrovskaya, A. A. Biochemistry 2000, 65, 95.
2. Nielsen, D.; Maare, C.; Skovsgaard, T. Gen. Pharmacol. 1996, 27, 251.
3. Lowenthal, R. M.; Eaton, K. Hematol. Oncol. Clin. North Am. 1996, 10, 967.
4. Huang, P. S.; Oliff, A. Curr. Opin. Genet. Dev. 2001, 11, 104.
5. Mittal, S.; Song, X.; Vig, B. S.; Landowski, C. P.; Kim, I.; Hilfinger, J. M.; Amidon,
G. L. Mol. Pharm. 2005, 2, 37.
In this study, we have synthesized the proline prodrug of meth-
otrexate using solid-phase peptide synthesis method. To specifi-
cally synthesize the prodrug coupling the amino group of proline
6. Lee, V. H. L. Eur. J. Pharm. Sci. 2000, 11, S41.
7. Sadee, W.; Drubbisch, V.; Amidon, G. L. Pharm. Res. 1995, 12, 1823.
8. Siotnak, F.; Burchall, J.; Ensminger, W.; Montgomery, J. Folate Antagonists Ther.
Agents 1984, 2, 166.
9. Siotnak, F.; Burchall, J.; Ensminger, W.; Montgomery, J. Folate Antagonists Ther.
Agents 1984, 2, 133.
10. Lindgren, M.; Rosenthal-Aizman, K.; Saar, K.; Eiríksdóttir, E.; Jiang, Y.; Sassian,
M.; Östlund, P.; Hällbrink, M.; Langel, Ü. Biochem. Pharmacol. 2006, 71, 416.
11. Chrzanowski, K.; Palka, J. Folia Histochem. Cytobiol. 2001, 39, 209.
12. Chrzanowski, K.; Bielawska, A.; Palka, J. Farmaco 2003, 58, 1113.
13. Chrzanowski, K.; Bielawska, A.; Palka, J. Rocz. Akad. Med. Bialymst. 1998, 43,
201.
and
-protected glutamate. The acid-labile t-butyl protecting group of
glutamate was easily removed when the prodrug is cleaved off
from Wang resin in acidic condition. The -coupling is preferred
over -coupling to mimic natural dipeptide substrate. It is also pro-
a-carboxylic acid group of methotrexate, we utilized the
c
a
c
posed that
DHFR than
c
a
-carboxylic acid contribute more to the binding with
-carboxylic acid. This would increase the possibility
14. Fmoc–proline–Wang resin (200 mg, 0.1 mmol) was added in a reaction tube
with filter and washed three times with 5 ml of 20% piperidine in DMF solution
for 10 min to remove protecting group Fmoc. Then it was washed with DMF
solution three times. HOBT (40.53 mg, 0.3 mmol), HBTU (113.79 mg,
0.3 mmol), Fmoc-Glu(OtBu)-OH (127.5 mg, 0.3 mmol) and triethylamine
(TEA) (30.57 mg, 0.3 mmol) in DMF solution were added to react for 1 h on
shaker. After draining the solution, the resin was washed with DMF three times
and 20% piperidine in DMF three times, respectively, to remove Fmoc. HOBT
(40.53 mg, 0.3 mmol) and HBTU (113.79 mg, 0.3 mmol), 4-[N-(2,4-diamino-6-
pteridinylmethyl)-N-methyl amino] benzoic acid hemi hydrochloride hydrate
(127.5 mg, 0.3 mmol) and triethylamine (TEA) (30.57 mg, 0.3 mmol) in DMF
solution were added to react for overnight on shaker. After draining the
solution, the resin was washed with DMF three times and methanol three
times, respectively, and dried in vacuum. After the resin was completely dry,
5 ml of mixture (95% trifluoroacetic acid, 2.5% water, and 2.5%
triisopropylsilane) was added to react for 90 min on shaker. The solution was
filtered into a 15 ml falcon tube and blew with nitrogen gas until the volume
for Pro-MTX to exhibit cytotoxicity without being converted to
methotrexate itself.
Pro-MTX showed good chemical stability in pH 7.4 PBS buffer
without the presence of prolidase or other hydrolyzing enzymes
(data not shown). In hydrolysis studies with prolidase, Pro-MTX
was found to be a modest substrate. Caco-2 and HeLa cell homog-
enates were also capable of hydrolyzing Pro-MTX to release pro-
line. This suggests that enzymes contribute significantly to the
hydrolysis of the Pro-MTX. In addition to prolidase, other enzymes
may also play important roles in hydrolysis of Pro-MTX. For exam-
ple, carboxypeptidase A was found to be able to activate a number
of amino acid methotrexate prodrugs effectively.25 Other possible
enzymes include prolylcarboxypeptidase and carboxypeptidase
P.26 Although prolidase is thought to require a dipeptide substrate
with small molecular weight,27 it is found that prolidase may have
a broader substrate specificity than thought previously.13,21
Proline analog of methotrexate evokes weaker cytotoxicity than
methotrexate in Caco-2 and MCF-7 Cells. This is consistent with
other researcher’s results.10 The intrinsic cytotoxicity of metho-
trexate prodrugs was lower compared to that of the parent drug
was reduced to ꢀ200
ll. Ten milliliters of cold diethylether was added to
obtain yellow precipitation. The suspension was centrifuged for 10 min at
2000 rpm and the supernatant was removed to obtain dry powder as Pro-MTX.
15. Spectroscopic data and yield of Pro-MTX:
Pro-MTX (1): yield, 45%; percent purity, 95%; 1H NMR (DMSO): 1.70–1.95 (m,
4H, CH2CH2COOH, proline CH2CH2), 2.11 (m, 2H, proline CH2CH2), 3.13 (m, 2H,
CH2CH2COOH), 3.57 (m, 2H, proline N–CH2), 3.27 (s, 3H, N–CH3), 4.15 (m, 1H,
a-H), 4.34 (m, 1H, a-H), 4.76 (s, 2H, CH2-N–CH3), 6.70 (d, 2H, CHbenzine–C–N),
7.65 (d, 2H, CHbenzine–C–CO), 8.61 (s, 1H, pteroyl ring); ESI-MS: 552 (M+H)+.
16. All cell lines were cultured at 37 °C with 5% CO2 and 90% relative humidity.
MCF-7 and MDA-MB-231 cells were cultured in 90% minimum essential
medium (MEM) with 10% FBS. Caco-2 cells were cultured in 80% minimum
essential medium (MEM) with 20% FBS. HeLa cells were cultured in 90% DMEM
with 10% FBS. Split confluent culture 1:4 to 1:6 every 3–5 days using trypsin/
EDTA. After trypsinization, the cells were washed three times with pH 7.4 PBS
buffer and re-suspended in pH 7.4 PBS (10 mM). To prepare cell homogenate,
add 1% Triton-X 100 in PBS solution and vortex vigorously. The cell suspension
was centrifuged at 18,000 rpm for 30 min at 4 °C. The supernatant was used in
hydrolysis study and to determine protein content. Total protein was
quantified with the BioRad Protein Assay using bovine serum albumin as
even we purposely synthesized the a-coupling prodrug to improve
the chance to bind to DHFR. Resistance to chemotherapy limits the
effectiveness of anticancer drugs. For MDA-MB-231 cell line, which
is resistant to methotrexate due to compromised RFC, Pro-MTX im-
proved cytotoxicity significantly compared to methotrexate itself.
This suggests proline prodrug of methotrexate may overcome the
resistance of MDA-MB-231 cell. The increased cytotoxicity might
be due to two mechanisms. First, Pro-MTX could be more effec-
tively transported into the MDA-MB-231 cells. Although the exact
mechanism is not known, transporters may play important roles
here. It has been proven that many amino acid coupled nucleosides
including valcyclovir and valganciclovir could be transported into
cells as substrates of peptide or nucleoside transporters.28,29 Lind-
gren et al. discovered that methotrexate can be successfully deliv-
ered by the new cell penetrating peptide into MDA-MB-231 cell
line to improve cytotoxicity.10 Secondly, it is also possible that
higher prolidase activity in MDA-MB-231 cell line (threefold higher
than that in non-methotrexate-resistant MCF-7 cell) contributes to
release of the more cytotoxic methotrexate in the cells. The combi-
nation of both mechanisms is also possible to be responsible for
the observed results. Further studies such as uptake study using
cell lines over-expressing peptide or nucleoside transporters could
be carried out to elucidate the exact mechanisms.
standard. The protein content was adjusted to ꢀ1000
lg/ml by appropriate
dilutions before being used in hydrolysis studies.
17. Myara, I.; Charpentier, C.; Lemonnier, A. Clin. Chim. Acta 1982, 125, 193.
18. For cytotoxicity study, 500 of Caco-2, MCF-7, or MDA-MB-231 cell in
ll
medium was cultured in 24 well plates. Generally, cells should be seeded at a
density of 5 Â 104 cells per well. It was allowed to grow for 48 h at 37 °C and 5%
CO2. Culture media was replaced by fresh media in each well. The synthesized
prodrugs and methotrexate with different concentrations were added and
incubated for 24 h. Hundred microliters of XTT mixture was added in each well
and incubate for 4 h. Growth medium alone was used as controls. UV
absorbance of the samples was measured at 490 nm.
19. Chrzanowski, K.; Bielawska, A.; Bielawski, K.; Woczynski, S.; Palka, J. Farmaco
2001, 56, 701.
20. Bielawski, K.; Bielawska, A.; Słodownik, T.; Bołkun-Skórnicka, U.; Muszyn´ ska,
A. Pharmacol. Rep. 2008, 60, 171.
21. Bielawska, A.; Bielawski, K.; Pałka, J. Rocz. Akad. Med. Bialymst. 1997, 42, 148.
22. Mittal, S.; Tsume, Y.; Landowski, C. P.; Lee, K. D.; Hilfinger, J. M.; Amidon, G. L.
Eur. J. Pharm. Biopharm. 2007, 67, 752.
23. Mittal, S.; Song, X.; Vig, B. S.; Amidon, G. L. Pharm. Res. 2007, 24, 1290.
24. Worm, J.; Kirkin, A. F.; Dzhandzhugazyan, K. N.; Guldberg, P. J. Biol. Chem. 2001,
276, 39990.
25. Kuefner, U.; Lohrmann, U.; Montejano, Y. D.; Vitols, K. S.; Huennekens, F. M.
Biochemistry 1989, 28, 2288.
In this study, we designed and synthesized proline prodrug of
methotrexate (Pro-MTX) to target methotrexate resistant MDA-
MB-231 which has higher prolidase activity and proline prodrug

5112
Z. Wu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5108–5112
26. Walter, R.; Simmons, W. H.; Yoshimoto, T. Mol. Cell. Biochem. 1980, 18, 111.
27. Bai, J. P.; Hu, M.; Subramanian, P.; Mosberg, H. I.; Amidon, G. L. J. Pharm. Sci.
1992, 81, 113.
28. Beutner, K. R. Antiviral Res. 1995, 28, 281.
29. Cocohoba, J. M.; McNicholl, I. R. Ann. Pharmacother. 2002, 36, 1075.