Methotrexate γ-hydroxamate derivatives as potential dual target antitumor drugs

Article abstract of DOI:10.1016/j.bmc.2006.11.017

A series of new aminopteroyl-based hydroxamate derivatives were synthesized and tested in vitro in cell culture models as potential dual target drugs. These compounds were designed to target two families of enzymes, matrix metalloproteinases (MMP) and a f

Full text of DOI:10.1016/j.bmc.2006.11.017

Bioorganic & Medicinal Chemistry 15 (2007) 1266–1274
Methotrexate c-hydroxamate derivatives as potential dual target
antitumor drugs
a,
a
b
b
*
´
M. Amelia Santos, Eva A. Enyedy, Elisa Nuti, Armando Rossello,
Natalia I. Krupenkoc and Sergey A. Krupenkoc
aCentro de Quı´mica Estrutural, Instituto Superior Te´cnico, 1049-001 Lisboa, Portugal
b
`
Universita degli Studi di Pisa, Dipartimento di Scienze Farmaceutiche Via Bonanno 6, 56126 Pisa, Italy
cDepartment of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA
Received 16 August 2006; revised 2 November 2006; accepted 10 November 2006
Available online 14 November 2006
Abstract—A series of new aminopteroyl-based hydroxamate derivatives were synthesized and tested in vitro in cell culture models as
potential dual target drugs. These compounds were designed to target two families of enzymes, matrix metalloproteinases (MMP)
and a folate enzyme, dihydrofolate reductase (DHFR). These enzymes are the components of two unrelated cellular pathways and
they are often over-expressed in metastasizing tumors. In addition to the synthesis and full structural characterization of the hybrid
molecules, we describe their inhibitory activities against a series of MMPs (MMP-2, MMP-7, MMP-9, MMP-14) and DHFR, as
well as their antiproliferative activity in three cancer cell lines. The new hydroxamate derivatives of MTX proved to be effective
inhibitors of MMPs and DHFR in the micromolar and nanomolar range, respectively. Furthermore, they showed strong antipro-
liferative activity against A549 cells (non-small cell lung carcinoma), and PPC-1 and Tsu-Pr1 prostate cancer cell lines. Therefore,
based on the present results, these bi-functional drugs may be good candidates to target specific tumors in animal models due to
potential combined effects on two pathways crucial for tumor development.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
tion.4,5 Therefore, inhibition of these enzymes is expect-
ed to slow or prevent the progression of these diseases.
A large number of synthetic MMP inhibitors (MMPIs)
have been developed as potential therapeutics.6–10 Since
all the MMPs possess a catalytic zinc ion in their active
site, MMP inhibitors contain a zinc-binding group
(ZBG) linked to different scaffolds to assure strong inter-
actions within the cofactor-binding region of the en-
zymes. The most developed class of MMPIs contains
the hydroxamate as the ZBG group.8 Several potent
MMPIs have been tested for tumor treatment, but at
present none of them are approved as drugs. Among
the 28 MMPs characterized so far, the gelatinases
(MMP-2, MMP-9), the matrilysin (MMP-7), and the
Membrane-Type 1-metalloproteinase (MMP-14) are
considered to play the most crucial role in promoting tu-
mor progression, facilitating metastatic dispersion and
angiogenesis.5,6,10 Therefore, specific inhibitors of these
MMPs are of high interest as potential anticancer
drugs.11
Zinc-containing enzymes, including carboxypeptidase A
(CPA), histone deacetylases (HDACs), tumor necrosis
factor a-convertase (TACE), and matrix metalloprotein-
ases (MMPs), are attractive therapeutic targets in treat-
ment of numerous diseases.1 Among these enzymes,
MMPs, a family of secreted or transmembrane proteas-
es, are of particular interest due to their role in degrada-
tion of the extracellular matrix (ECM). MMPs are
tightly regulated at multiple levels, including regulation
of their activity by the endogenous tissue inhibitors of
MMPs (TIMPs).2,3 Under normal physiological condi-
tions these enzymes are not highly expressed. Uncon-
trolled overexpression of MMPs can lead to tissue
degradation and promote a variety of diseases that re-
quire tissue remodeling, including: arthritis, tumor
metastasis, multiple sclerosis, and periodontal degrada-
Keywords: Antifolates; Methotrexate-hydroxamate derivatives; Dual
drugs; MMP inhibitors; DHFR inhibitors.
*
Antifolates are another class of molecules that have
been used for many years in cancer chemotherapy.12–18
Corresponding author. Tel.: +351 21 8419273; fax: +351 21
8464455; e-mail: masantos@ist.utl.pt
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.11.017

M. A. Santos et al. / Bioorg. Med. Chem. 15 (2007) 1266–1274
1267
They are the folate analogs that inhibit key folate en-
zymes, namely thymidylate synthase (TS) and dihydro-
folate reductase (DHFR).12 This inhibition induces
cytotoxic effects by the ultimate suppression of de novo
biosynthesis of purine nucleotides and thymidylate.18
The antifolate methotrexate (4-amino-4-deoxy-N10-
methylpteroylglutamic acid, MTX) is one of the most
widely used drugs in medical oncology for treatment
of several types of cancer.19 It is also used in the treat-
ment of inflammatory diseases such as rheumatoid
arthritis.20 MTX differs from folic acid by two substitu-
tions: (i) a hydroxyl is replaced by an amino group at C4
position of the pterine ring; (ii) a methyl group is added
to N10 position (Fig. 1). Such a ‘minor’ difference con-
verts the DHFR substrate into a tight-binding inhibi-
tor.13,21 MTX is effectively transported across the cell
membrane by a carrier-mediated mechanism.17,21,22
Upon entering the cell, it undergoes polyglutamylation
that decreases its efflux and enhances inhibitory
effects.12,13,21
different types of cancers.29–31 In this strategy, the use
of drugs targeting different, often unrelated, pathways,
with expectation of synergistic effects, becomes an ap-
proach of increasing interest. As a long-standing chemo-
therapeutic, MTX has been successfully used in
combination with other drugs for quite some time.32–34
In this regard, MMP inhibitors are not well studied
although their synergistic effects with other anticancer
drug have been observed in animal models.35 Interest-
ingly, recent phase I studies evaluated effects of an
MMP inhibitor in combination with 5-fluorouracil (the
TS inhibitor) and leucovorin in patients with advanced
solid tumors.36 Further development of combination
therapy involves the use of multifunctional drugs, when
one chemical compound can hit two or more targets.37
Such approach has high potential to improve therapeu-
tic efficacy. In our studies, we pursued the development
of a new type of double-target drugs by combining two
structurally unrelated inhibitory functions (targeting fo-
late pathways and MMPs) in a single molecule. Since
MMP-dependent tissue remodeling and folate requiring
biosynthetic pathways are crucial for tumor progression,
simultaneous management of both types of pathways,
by a single drug, could be highly beneficial. MTX still
remains an effective drug in cancer treatment, therefore
in our design strategy it was selected as the antifolate
compound to be modified into a series of hydroxamate
derivatives. The synthesized derivatives were tested for
their inhibitory activity toward DHFR and several
MMPs as well as for their antiproliferative activity in
cancer cell lines.
Efficacy of MTX can be limited by acquired resistance
that occurs through multiple mechanisms including: de-
creased MTX transport, diminished polyglutamylation
of the drug, overexpression of its main target, DHFR,
or expression of MTX-resistant DHFR mu-
tants.12,17,21,23 To produce more potent drugs that can
circumvent resistance to MTX, novel antifolates have
been developed, including a new generation of multitar-
get drugs.24–28 Pemetrexed, a multitarget drug, targeting
DHFR and TS, was the first antifolate approved by the
US FDA (February, 2004) since the introduction of
MTX more than 50 years ago.26
2. Molecular design
In recent years, together with development of new
drugs, significant efforts have been directed toward the
development of combination therapies in treatment of
The design of the compounds, reported in the present
paper, is based on the addition of extra functional moi-
Methotrexate (MTX) 1a X=OH
2a X=NHOH
O
COOH
OH
O
O
NH
NH2
N
N
H
N
N
X
N
N
3a X=
4a X=
N
H
H2N
OH
NH
O
N
O
COOH
Folic acid 1b X=OH
2b X=NHOH
O
OH
NH
O
N
H
O
N
X
HN
H2N
N
H
3b X=
4b X=
N
H
N
N
OH
NH
O
N
Figure 1. Structures of MTX, folic acid, and their hydroxamic acid derivatives 2a,b–4a,b.

1268
M. A. Santos et al. / Bioorg. Med. Chem. 15 (2007) 1266–1274
ety to MTX molecule. In an attempt to improve selectiv-
ity and decrease toxicity, numerous derivatives of MTX
have been synthesized.38–40 In general, only the c-car-
boxyl has been modified because the free a-carboxyl
group contributes significantly to the binding of MTX
to DHFR. In particular, incorporation of non-polar
groups such as alkylamino-acids or alkylic esters, and
monohydroxamic group, at the c-position has been per-
formed.40 A monohydroxamate derivative of MTX was
found to be moderately toxic to human and mouse leu-
kemia cells.41 Thus, the main goal of the present studies
was an attempt to improve MTX as anticancer drug by
adding an additional function to MTX molecule.
mic acid through condensation with free hydroxylamine.
All reactions were performed in dry DMF under nitro-
gen atmosphere to exclude moisture. All synthesized
compounds were characterized by NMR spectroscopy.
3.2. MMPs inhibition
The synthesized compounds and the reference drug CGS
27023A (used as a control) were tested as inhibitors of
MMPs often overexpressed in tumors (MMP-2, MMP-
7, MMP-9, and MMP-14).6 The hydroxamic acid deriv-
atives of folic acid and MTX showed inhibitory effects
on studied MMPs in the micromolar range (Table 1).
Under the same experimental conditions, GGS 27023A
resulted in a much more potent inhibition with IC50 in
the nanomolar range.
The MTX-derivatives developed herein should be effi-
cient inhibitors of specific MMPs in addition to their
DHFR inhibition capabilities. Accordingly, the MTX
structural elements, responsible for the interaction with
active site of DHFR, were kept, while the hydroxamate
moiety, a strong zinc-binding group (ZBG), was intro-
duced at the c-position of MTX glutamate (by replace-
ment of the c-COOH by c-CONHOH) to enable the
MMPs’ inhibition. The efficacy of a MMP inhibition de-
pends on two factors: (i) the ability to bind the catalytic
zinc ion; (ii) the aptitude to form contacts with the en-
zyme subsites (via hydrogen and van der Waals
bonds).6,7 To enhance the capability of the inhibitors
to bind to MMPs, monoamino acid spacers, containing
hydrophobic residues, were introduced between the
hydroxamate group and the core molecule.
Interestingly, in the case of folic acid derivatives, the
insertion of an amino acid spacer between the folate
molecule and the hydroxamate group improved inhibi-
tory potency as compared with the folate hydroxamate
2b (Table 1). In particular, introduction of the phenylal-
anine spacer (3b) improved the inhibitory effects, espe-
cially against MMP-7 (IC50 = 15 lM) and MMP-9
(IC50 = 22 lM). Substitution of the phenylalanine by
the proline spacer (4b) resulted in a reduction of the
inhibitory effects on MMP-2 and MMP-7, although
the inhibitory effects of these derivatives against
MMP-9 (IC50 = 30 lM) and MMP-14 (IC50 = 100 lM)
were similar. In the case of MTX hydroxamic acid deriv-
atives, the introduction of an amino acid spacer also
enhances MMP inhibitory potency. In particular, 3a
derivative shows IC50 values ranging from 15 lM on
MMP-9 to 70 lM on MMP-14. Overall, enhanced
inhibitory effects against the tested MMPs were ob-
served when a flexible lipophilic amino acid spacer
(phenylalanine-hydroxamate) was added between the
core molecule and the hydroxamic group.
Folic acid derivatives, analogous to the MTX
c-hydroxamate derivatives, were also synthesized and
studied as the control compounds. Thus, the following
derivatives of MTX and folic acid were synthesized
and tested (see Fig. 1): methotrexate c-L-phen-
ylalaninehydroxamic acid (3a), methotrexate c-L-pro-
linehydroxamic acid (4a), methotrexate c-hydroxamic
acid (2a); folate c-L-phenylalaninehydroxamic acid
(3b), folate c-L-prolinehydroxamic acid (4b), folate
c-hydroxamic acid (2b).
3.3. DHFR inhibition
The synthesized derivatives of MTX and folic acid were
evaluated for the ability to inhibit activity of DHFR, the
MTX target. DHFR is capable of catalyzing two reac-
tions in folate cycle, conversion of folic acid to dihydro-
3. Results and discussion
3.1. Chemistry
folate
and
conversion
of
dihydrofolate
to
tetrahydrofolate.42 The DHFR-catalyzed conversion of
dihydrofolate to tetrahydrofolate is a much faster reac-
tion than conversion of folic acid to dihydrofolate and it
is viewed as a major metabolic function of DHFR. The
other reaction, however, is also important if the source
of folate is folic acid. In our cell culture experiments
(see Experimental section) the cells were supplemented
with folic acid as the only source of folate. The absorbed
folic acid should be first converted to dihydrofolate in a
DHFR-catalyzed reaction; accordingly, inhibition of
cellular growth in our experiments is likely to be related
to the inhibition of this reaction since this is the first and
required step for the entry of folic acid into the reduced
folate pool. Therefore, both folic acid and dihydrofolic
acid were used as a substrate to evaluate DHFR inhibi-
tion in vitro. Overall, all tested MTX-based compounds
produced similar inhibitory effects on both reactions.
Commercial preparations of MTX and folic acid were
used in our syntheses (Scheme 1). The first step was acti-
vation of the c-carboxylic group of MTX or folic acid
that was achieved by using stoichiometric amount of
TBTU in the presence of N-methyl-morpholine. Prefer-
ential modification of the c-carboxylic group over the
a-carboxylic group was due to hydrogen bonding of
the latter to the neighboring amide group and limited
access of the bulky modifying agent to the a-position.
Subsequent condensation with the amino acid molecules
(L-Phe, L-Pro) or the free hydroxylamine (Scheme 1)
yielded the corresponding MTX/folic acid peptide deriv-
atives or the simple hydroxamate derivatives. The syn-
thesis of the amino acid-hydroxamate derivatives
involved an additional step to convert the carboxylic
group of the amino acid to the corresponding hydroxa-

M. A. Santos et al. / Bioorg. Med. Chem. 15 (2007) 1266–1274
COOH
1269
O
O
R
N
H
N
N
OH
N
N
1a (MTX)
1b Folic acid
R1
a: R=NH2; R1=Me
b: R=OH; R1=H
H2N
N
i, ii
O
COOH
O
NHOH
R
N
H
N
N
N
R1
H2N
N
N
i, iii
i, iii
2a,b
O
COOH
O
R
N
H
N
N
NH
COOH
N
N
Ph
R1
O
COOH
H2N
N
O
5a,b
R
N
H
N
N
N
COOH
N
N
R1
6a,b
H2N
N
i, ii
i, ii
O
COOH
O
R
N
H
N
N
NH
CONHOH
N
N
Ph
R1
O
COOH
H2N
N
O
3a,b
R
N
H
N
N
N
CONHOH
N
N
R1
H2N
N
4a,b
Scheme 1. Synthesis of the c-hydroxamic acid derivatives of MTX and folic acid. Reagents and conditions: (i) TBTU, NMM, 0 °C, dry DMF; (ii)
NH2OH, 0 °C, dry DMF; (iii) aminoacid (L-Phe, L-Pro), 0 °C, dry DMF.
Table 1. Inhibitory activity (IC50 lM or % inhibition at 100 lM) of a set of c-hydroxamate derivatives of the folic acid (2b–4b), of the MTX (2a–4a),
and a reference drug CGS 27023A toward the selected MMPs
Compound
MMP-2
MMP-7
MMP-9
MMP-14
2b
3b
66%
65 (2708)a
58%
53%
15 (150)a
54%
55%
22 (4400)a
30
42%
80 (3478)a
100
4b
2a
67%
100
61%
48%
3a
4a
60 (2500)a
56%
0.024
44 (440)a
43%
0.1
15 (3000)a
61%
0.005
70 (3043)a
47%
0.023
CGS 27023A
a IC50-inhibitor/IC50-CGS 27023A ratios.
These experiments revealed that the derivatives of MTX
possessed strong inhibitory effects toward DHFR with
IC50 in a hundred nanomolar range (Table 2). In con-
trast, the folate analogs did not show notable inhibition
at concentration up to 100 lM (Table 2). Compared to
MTX, however, the hydroxamate derivatives of MTX
displayed lower inhibitory effects on DHFR activity.
We explain this by decreasing affinity of DHFR to the
derivatives, due to sterical hindrance of the substitute
group in the active site of the enzyme. Indeed, 3a, the
derivative with the most bulky spacer, showed the low-
est inhibitory effect. In addition, at neutral pH the car-
boxylate is negatively charged, while the hydroxamate
group has no charge. Thus, the modification of the car-

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M. A. Santos et al. / Bioorg. Med. Chem. 15 (2007) 1266–1274
Table 2. Antiproliferative data, IC50 (lM), of c-hydroxamate derivatives of folic acid and MTX on cultured cancer cells (A549, PPC-1, Tsu-Pr1);
inhibitory data of MTX c-hydroxamate derivatives, IC50 (nM), against dihydrofolate reductase (DHFR)
Compound
Antiproliferative activitya IC50 (lM)
DHFRb IC50 (nM)
A549
PPC-1
Tsu-Pr1
Folic acid
DHF
2b
3b
100
110
130
6.0
6.5
7.5
0.1
80
70
120
150
80
NDc
ND
ND
170
250
130
35
ND
ND
ND
70
4b
2a
120
2.0
4.8
1.8
0.1
2.0
2.0
2.3
0.1
3a
4a
170
120
9
MTX
a Average of two experiments performed in triplicate is shown except for MTX-derivatives; average of three experiments performed in triplicate is
shown for MTX-derivatives.
b DHFR was assayed using as a substrate folic acid or dihydrofolic acid (DHF).
c ND, non-detectable (no effects on DHFR activity were observed at up to 100 lM inhibitor concentration).
boxylic moiety of glutamic acid eliminates the negatively
charged group that could contribute to binding with
DHFR.
Table 3. IC50 (lM) of MTX hydroxamate derivatives in cultured
cancer cells (A549, PPC-1, Tsu-Pr1) grown on folate-depleted media
Compound
A549
PPC-1
Tsu-Pr1
2a
3a
0.1
0.5
0.5
0.02
0.05
0.5
0.01
0.12
0.1
3.4. Cell proliferation assays
4a
MTX
0.2
0.01
The antiproliferative effects of the folate and methotrex-
ate-hydroxamate derivatives have been evaluated on
three cell lines, A549 (lung carcinoma), Tsu-Pr1 and
PPC-1 (prostate carcinomas). Effects of these com-
pounds were compared to the antiproliferative effects
of MTX as a control. Cell viability was assessed by
MTT cell proliferation assay as described elsewhere.43
For all three tested cell lines, we have observed that
derivatives of MTX were much stronger inhibitors of
proliferation than folate derivatives (Table 2). However,
none of the tested inhibitors was as effective as MTX it-
self: IC50 for MTX was about 0.1 lM, while IC50 for all
MTX-derivatives were in a micromolar range. Although
some antiproliferative effects of the folate derivatives
were observed at higher concentrations (in the range
near 10ꢀ4 M), this could be explained by the simple
competition with media folate for the folate transport-
ers. Since concentration of folate in media (2.2 lM) is
much lower than the effective concentrations of the fo-
late derivatives, these derivatives could interfere with fo-
late uptake. In the cell, folate derivatives apparently
cannot substitute for folate in folate-required reactions,
most likely due to loss of polyglutamylation.
0.008
Average of two experiments performed in triplicate is shown.
Compound 2a, that has the least bulky substituent,
was the most effective of MTX-derivatives in inhibition
of proliferation.
Overall, among the synthesized compounds, only MTX
hydroxamate derivatives, but not folic acid derivatives,
demonstrated strong inhibitory effects (nanomolar
range) against DHFR. This suggests that the growth
suppressor effects resulted from DHFR inhibition and
should be attributed to MTX component. The enhance-
ment of the suppressor effects of MTX hydroxamate
derivatives in folate-depleted media further suggests that
these effects are associated with the folate metabolism.
More pronounced reduction of antiproliferative activity
of MTX derivatives, as compared to MTX itself, than
the decrease in DHFR inhibition could be an indication
of diminished cellular transport of the synthesized deriv-
atives. Alternatively, it could be due to the loss of poly-
glutamylation inside the cell: it is well known that
polyglutamylated forms of MTX are stronger inhibitors
than MTX monoglutamate.21
In another set of experiments we have tested effects of
MTX-derivatives on proliferation of cells grown on
folate-depleted media. Regular medium is supplemented
with 2.2 lM folic acid that is a high folate concentra-
tion. In experiments with lower folate supplementation,
cells were grown on folate-free media supplemented with
20 nM leucovorin (5-formyltetrahydrofolate). Cells
grow very slowly in low folic acid media, while leucovo-
rin, a reduced folate, can support normal cell growth at
very low concentrations.44 At low folate supplementa-
tion, antiproliferative effects of MTX-derivatives were
more profound and closer to the effects of MTX itself
(Table 3). Thus, IC50 were in the 10ꢀ8–10ꢀ7 M range
for all three cell lines. Among the three cell lines, PPC-
1 cells were the most sensitive to MTX and MTX-based
inhibitors, while A549 cells were the most resistant.
4. Conclusions
c-Monohydroxamate derivatives of MTX, a novel type
of prospective pharmacological agents with dual target
capability, have been developed and tested. Our results
demonstrated that the synthesized derivatives retain
capability to inhibit two independent targets of metasta-
sizing tumors, matrix metalloproteinases (MMPs) and
dihydrofolate reductase (DHFR). In general, these stud-
ies demonstrated the feasibility of our approach in the
development of multitarget drugs capable to inhibit
two different enzyme systems. In terms of chemothera-

M. A. Santos et al. / Bioorg. Med. Chem. 15 (2007) 1266–1274
1271
py, combination of two inhibitory determinants in one
5.2. Synthesis of the compounds
molecule opens an opportunity to target simultaneously
proliferation of primary tumor cells and tumor inva-
siveness. Thus, the synthesized compounds could be
good candidates for testing on experimental tumors in
animal models. In general, the precautions should be
taken in development of MTX-based dual target inhib-
itors: the preferential binding of MTX to DHFR, due
to an extremely high affinity, could consume most of
the intracellular inhibitor making it unavailable to tar-
get other enzymes. However, in our case compartmen-
talization of the selected targets allows avoiding this
problem. Indeed, MMPs are extracellular enzymes,
while DHFR is intracellular. Therefore, it could be
expected that the synthesized compounds upon delivery
to the tumors will inhibit MMPs first, preventing
metastasis, and then, after translocation into tumor
cells, will further inhibit proliferation by targeting
DHFR.
5.2.1. Folate c-hydroxamic acid (2b). To a solution of fo-
lic acid (0.85 g, 2 mmol) in dry DMF (40 mL) under
nitrogen was added N-methylmorpholine (0.45 mL,
4 mmol) at room temperature and then TBTU (0.65 g,
2 mmol) under cooling with an ice-water bath. This
reaction mixture was stirred for 45 min at 0 °C. To a
solution of hydroxylamine hydrochloride (0.42 g,
6 mmol) in dry DMF (10 mL) under nitrogen, contain-
ing activated molecular sieves (0.4 nm, Riedel-de Hae¨n),
was added KOH (0.34 g, 6 mmol). The resulting mixture
was stirred for 30 min and the inorganic solids were fil-
tered off from the solution. The first solution was slowly
added to the second one and the mixture was stirred for
5 h at 0 °C and then it was kept at 0 °C for 12 h. The sol-
vent was evaporated under vacuum, the solid residue
was diluted into CH2Cl2 and recrystallized from dry eth-
anol. The resulting solid was washed with ethanol/dieth-
yl ether to give 2b (0.64 g, 70%) as a crystalline solid, mp
1
230–240 °C (decomposition). H NMR (D2O, pD = ca.
5. Experimental
9) d: 8.66 (1H, s, CH@N), 6.63 (1H, d, CH@C–NH),
7.59 (2H, d, CH@C–C@O), 4.29 (1H, s, CH–COOH),
4.47 (2H, s, CH2–NH), 2.08 (2H, d, CH2–CH), 2.29
(2H, t, CH2–C@O); 13C NMR (DEPT) (D2O, pD = ca.
9) d: 150 (CH@N), 115 (CH@C–NH), 132 (CH@
C–C@O), 58.4 (CH–COOH), 48.5 (CH2–NH), 36.8
(CH2–CH), 31.0 (CH2–C@O); IR: 1513 cmꢀ1 (C@O,
new amide bond); HRMS (ESI) calcd for (M+1)
457.15786; found: 457.15803.
5.1. General methods
All reagents were of commercial quality and used with-
out further purification. Folic acid, methotrexate,
O-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium-tet-
rafluoroborate (TBTU), L-phenylalanine (L-Phe), L-pro-
line (L-Pro), N-methylmorpholine (redistilled), and
hydroxylamine hydrochloride were purchased from Sig-
ma–Aldrich. The solvents were purchased from Acros
Organics or Merck; whenever necessary, they were puri-
fied and dried according to standard methods.45 All the
moisture-sensitive reactions were performed under
nitrogen atmosphere. The chemical reactions were fol-
lowed by TLC using silica gel plates (G-60 F254, Merck).
A Bio-Rad Merlin FTS 3000 MX spectrometer was used
5.2.2. Folate c-L-phenylalanine (5b). A cooled solution
containing the activated folic acid (prepared as described
above) was slowly added to a water-ice cooled solution of
L-phenylalanine (0.33 g, 2 mmol) in dry DMF (10 mL)
and the reaction mixture was stirred for 2 h at 0 °C. After
being warmed up to room temperature, the reaction mix-
ture was evaporated in vacuo. The resulting residue was
diluted with CH2Cl2 and recrystallized from dry ethanol
to give 5b (88% yield) as a yellow solid. 1H NMR (D2O,
pD = ca. 9) d: 8.62 (1H, s, CH@N), 6.86 (2H, d,
CH@C–NH), 7.63 (2H, d, CH@C–C@O), 7.02 (5H, m,
C6H5), 4.47 (1H, s, CH–COOH), 4.31 (1H, m, CH–
CH2–C6H5), 4.62 (2H, s, CH2–NH), 2.01 (2H, d, CH2–
CH), 2.24 (2H, t, CH2–C@O), 3.02 (2H, m, CH2–C6H5);
13C NMR (DEPT) (D2O, pD = ca. 9), d: 150 (CH@N),
116 (CH@C–NH), 131 (CH@C–C@O), 132 (C6H5),
59.0 (CH–COOH), 57.0 (CH–CH2–C6H5), 48.3 (CH2–
NH), 34.1 (CH2–CH), 30.1 (CH2–C@O), 40.0 (CH2–
C6H5); IR: 1512 cmꢀ1 (C@O, new amide bond); MS
(FAB) m/z: 589 (M+1).
to record solid state IR spectra (KBr pellets). 1H and 13
C
NMR spectra were recorded on a Varian Unity 300 FT
NMR spectrometer (300 MHz) at 25 °C. If necessary,
assignment of the signals of the 13C NMR was con-
firmed by DEPT. Chemical shifts are reported in ppm
(d) from sodium 3-(trimethylsilyl)-[2,2,3,3-2H4]-propio-
nate as internal reference in D2O solutions. The follow-
ing abbreviations are used: d, doublet; s, singlet; t,
triplet; m, multiplet; br, broad. Mass spectra (FAB)
were obtained on a VG TRIO-2000 GCMS instrument.
High-resolution mass spectrometry (HRMS (ESI)) mea-
surements were performed on an APEX III FT-ICR MS
(Bruker Daltonics, Billerica, MA), equipped with a 7T
actively shielded magnet. Ionization was achieved by
an electrospray ionization source (Bruker Daltonics,
Billerica, MA), with a voltage between 1800 and
2200 V (to optimize ionization efficiency) applied to
the needle, and a counter voltage of 450 V applied to
the capillary. Samples were prepared by adding a spray
solution of 70:29.5:0.5 (v/v/v) CH3OH/water/formic acid
to a solution of the sample at a v/v ratio of 1–5% to give
the best signal-to-noise ratio. Data acquisition and pro-
cessing were performed using the XMASS software, ver-
sion 6.1.2 (Bruker Daltonics). Elemental analyses were
performed on a Fisons EA 1108 CHNF/O instrument.
5.2.3. Folate c-L-phenylalaninehydroxamic acid (3b). The
title compound was prepared from 5b according to the
procedure described for 2b. Yellow crystals (44% yield),
mp 255–260 °C (decomposition). 1H NMR (D2O,
pD = ca. 9) d: 8.59 (1H, s, CH@N), 6.93 (7H, m,
CH@C–NH, C6H5), 7.63 (2H, d, CH@C–C@O), 4.45
(1H, s, CH–COOH), 4.31 (1H, s, CH–CH2–C6H5),
4.59 (2H, s, CH2–NH), 1.98 (2H, d, CH2–CH), 2.25
(2H, t, CH2–C@O), 2.95 (2H, m, CH2–C6H5); 13C
NMR (DEPT) (D2O, pD = ca. 9) d: 151 (CH@N), 116
(CH@C–NH), 131 (CH@C–C@O), 132 (C6H5), 58.6

1272
M. A. Santos et al. / Bioorg. Med. Chem. 15 (2007) 1266–1274
(CH–COOH), 55.8 (CH–CH2–C6H5), 48.2 (CH2–NH),
31.9 (CH2–CH), 30.0 (CH2–C@O), 39.5 (CH2-C6H5);
IR: 1509 cmꢀ1 (C@O, new amide bond); Anal. Found:
C, 53.93; H, 4.94; N, 19.36%. C24H27N9O7Æ1.22 MeOH
requires: C, 53.65; H, 4.76; N, 20.11%; HRMS (ESI)
calcd for (M+1) 604.2263; found: 604.2278.
pD = ca. 9) d: 8.53 (1H, s, CH@N), 6.84 (2H, d,
CH@C–NH), 7.67 (2H, d, CH@C–C@O), 7.29 (5H,
C6H5), 4.26 (1H, s, CH–COOH), 4.18 (1H, s, CH–
CH2–C6H5), 4.74 (2H, s, CH2–NCH3), 2.04 (2H, d,
CH2–CH), 2.26 (2H, t, CH2–C@O), 2.87 (2H, m,
CH2–C6H5), 3.13 (3H, s, CH3); 13C NMR (DEPT)
(D2O, pD = ca. 9) d: 152 (CH@N), 115 (CH@C–NH),
130 (CH@C–C@O), 131 (C6H5), 58.3 (CH–COOH),
57.3 (CH–CH2–C6H5), 68.6 (CH2–NCH3), 34.7 (CH2–
CH), 29.5 (CH2–C@O), 41.5 (CH2–C6H5), 44.9 (CH3);
IR: 1511 cmꢀ1 (C@O, new amide bond); MS (FAB)
m/z: 602 (ꢁ100%) (M+1).
5.2.4. Folate c-L-proline (6b). The title compound was
prepared from folic acid and L-proline according to
the procedure described for 5b. Yellow crystals (81%
yield); 1H NMR (D2O, pD = ca. 9) d: 8.60 (1H, s,
CH@N), 6.84 (2H, d, CH@C–NH), 7.66 (2H, d,
CH@C–C@O), 4.27 (1H, s, CH–COOH), 4.19 (1H, s,
N–CH–CH2), 4.61 (2H, s, CH2–NH), 2.08 (6H, m,
CH2–CH, CH2–C@O, N–CH2–CH2–CH2), 3.56 (2H,
m, N–CH2–CH2–CH2), 2.47 (2H, m, N–CH2–CH2–
CH2); 13C NMR (DEPT) (D2O, pD = ca. 9) d: 150
(CH@N), 115 (CH@C–NH), 132 (CH@C–C@O), 65.4
(CH–COOH), 50.0 (N–CH–CH2), 47.4 (CH2–NH),
32.7 (CH2–CH), 29.6 (CH2–C@O), 39.5 (N–CH2–
CH2–CH2), 22.6 (N–CH2–CH2–CH2), 28.4 (N–CH2–
CH2–CH2); IR: 1513 cmꢀ1 (C@O, new amide bond);
MS (FAB) m/z: 539 (60%) (M+1).
5.2.8. Methotrexate c-L-phenylalaninehydroxamic acid
(3a). The title compound was prepared from 5a accord-
ing to the procedure described for 2b. Yellow crystals
1
(83% yield), mp 190–195 °C. H NMR (D2O, pD = ca.
9) d: 8.58 (1H, s, CH@N), 7.65 d (2H, d, CH@C–
C@O), 6.92 (7H, m, C6H5, CH@C–NH), 4.41 (1H, s,
CH–COOH), 4.27 (1H, s, CH–CH2–C6H5), 4.67 (2H,
s, CH2–NCH3), 1.96 (2H, d, CH2–CH), 2.17 (2H, t,
CH2–C@O), 2.82 (2H, m, CH2–C6H5), 3.22 (3H, s,
CH3); 13C NMR (DEPT) (D2O, pD = ca. 9) d: 152
(CH@N), 115 (CH@C–NH), 131 (CH@C–C@O), 132
(C6H5), 57.2 (CH–COOH), 56.4 (CH–CH2–C6H5), 68.7
(CH2–NCH3), 35.6 (CH2–CH), 29.8 (CH2–C@O), 39.0
(CH2–C6H5), 43.1 (CH3); IR: 1510 cmꢀ1 (C@O, new
amide bond); HRMS (ESI) calcd for (M+1) 617.2579;
found: 617.2582.
5.2.5. Folate c-L-proline-hydroxamic acid (4b). The title
compound was prepared from 6b according to the pro-
cedure described for 2b. Yellow crystals (37% yield),
mp 240–245 °C (decomposition). 1H NMR (D2O,
pD = ca. 9) d: 8.59 (1H, s, CH@N), 6.84 (2H, d,
CH@C–NH), 7.66 (2H, d, CH@C–C@O), 4.28 (1H, m,
CH–COOH), 4.19 (1H, s, N–CH–CH2), 4.59 (2H, s,
CH2–NH), 2.15 (6H, m, CH2–CH, CH2–C@O,
N–CH2–CH2–CH2), 3.64 (2H, m, N–CH2–CH2–CH2),
2.48 (2H, m, N–CH2–CH2–CH2); 13C NMR (DEPT)
(D2O, pD = ca. 9) d: 151 (CH@N), 115 (CH@C–NH),
131 (CH@C–C@O), 64.2 (CH–COOH), 50.2 (N–CH–
CH2), 47.9 (CH2–NH), 34.0 (CH2–CH), 32.1 (CH2–
C@O), 43.2 (N–CH2–CH2–CH2), 24.4 (N–CH2–CH2–
CH2), 27.0 (N–CH2–CH2–CH2); IR: 1512 cmꢀ1 (C@O,
new amide bond); Anal. Found: C, 50.95; H, 5.01; N,
20.94%. C28H29N9O7Æ0.64 HCl requires: C, 51.11; H,
5.42; N, 21.26%. HRMS (ESI) calcd for (M+1)
554.2106; found: 554.2103.
5.2.9. Methotrexate c-L-proline (6a). The title compound
was prepared from methotrexate and L-proline accord-
ing to the procedure described for 5b. Yellow crystals
1
(58% yield); H NMR (D2O, pD = ca. 9) d: 8.56 (1H,
s, CH@N), 6.86 (2H, d, CH@C–NH), 7.67 (2H, d,
CH@C–C@O), 4.40 (1H, s, CH–COOH), 4.23 (1H, s,
N–CH–CH2), 4.59 (2H, s, CH2–NCH3), 2.05 (6H, m,
CH2–CH, CH2–C@O, N–CH2–CH2–CH2), 3.51 (2H,
m, N–CH2–CH2–CH2), 2.45 (2H, m, N–CH2–CH2–
CH2), 3.15 (3H, s, CH3); 13C NMR (DEPT) (D2O,
pD = ca. 9) d: 152 (CH@N), 114 (CH@C–NH), 131
(CH@C–C@O), 57.4 (CH–COOH), 50.3 (N–CH–
CH2), 64.4 (CH2–NCH3), 33.5 (CH2–CH), 32.0 (CH2–
C@O), 41.3 (N–CH2–CH2–CH2), 26.6 (N–CH2–CH2–
CH2), 29.6 (N–CH2–CH2–CH2), 42.8 (CH3); IR:
1510 cmꢀ1 (C@O, new amide bond); MS (FAB) m/z:
552 (ꢁ100%) (M+1).
5.2.6. Methotrexate c-hydroxamic acid (2a). The title
compound was prepared from methotrexate according
to the procedure described for 2b. Recrystallization
from DMF provided yellow crystals with 62% yield,
mp 212–216 °C. 1H NMR (D2O, pD = ca. 9) d: 8.55
(1H, s, CH@N), 6.86 (2H, d, CH@C–NH), 7.69 (2H,
d, CH@C–C@O), 4.30 (1H, s, CH–COOH), 4.76 (2H,
CH2–NCH3), 2.08 (2H, d, CH2–CH), 2.25 (2H, t,
CH2–C@O), 3.14 (3H, s, CH3); 13C NMR (DEPT)
(D2O, pD = ca. 9) d: 152 (CH@N), 115 (CH@C–NH),
132 (CH@C–C@O), 58.1 (CH–COOH), 63.1 (CH2–
NCH3), 38.7 (CH2–CH), 31.0 (CH2–C@O), 42.5
(CH3); IR: 1514 cmꢀ1 (C@O, new amide bond); HRMS
(ESI) calcd for (M+1) 470.1895; found: 470.1907.
5.2.10. Methotrexate c-L-proline-hydroxamic acid (4a).
The title compound was prepared from 6a according to
the procedure described for 2b. Yellow crystals (56%
1
yield), mp 233–235 °C. H NMR (D2O, pD = ca. 9) d:
8.58 (1H, s, CH@N), 6.90 (2H, d, CH@C–NH), 7.66
(2H, d, CH@C–C@O), 4.40 (1H, m, CH–COOH), 4.21
(1H, s, N–CH–CH2), 4.61 (2H, s, CH2–NCH3), 1.99
(6H, m, CH2–CH, CH2–C@O, N–CH2–CH2– CH2),
3.48 (2H, m, N–CH2–CH2–CH2), 2.49 (2H, m, N–
CH2–CH2–CH2), 3.16 (3H, s, CH3); 13C NMR (DEPT)
(D2O, pD = ca. 9) d: 152 (CH@N), 115 (CH@C–NH),
131 (CH@C–C@O), 57.5 (CH–COOH), 49.3 (N–CH–
CH2), 68.4 (CH2–NCH3), 35.2 (CH2–CH), 31.1 (CH2–
C@O), 38.7 (N–CH2–CH2–CH2), 25.7 (N–CH2–CH2–
CH2), 27.9 (N–CH2–CH2–CH2), 42.2 (CH3); IR:
5.2.7. Methotrexate c-L-phenylalanine (5a). The title
compound was prepared from methotrexate and
L-phenylalanine according to the procedure described
for 5b. Yellow crystals (75% yield); 1H NMR (D2O,

M. A. Santos et al. / Bioorg. Med. Chem. 15 (2007) 1266–1274
1273
1509 cmꢀ1 (C@O, new amide bond); HRMS (ESI) calcd
for (M+1) 567.2423; found: 567.2437.
each MMP in the fluorimetric assay buffer (FAB): Tris
50 mM, pH 7.5, NaCl 150 mM, CaCl2 10 mM, Brij 35
0.05%, and DMSO 1%. The activated enzyme (final con-
centration 2.8 nM for MMP-2, 2.36 nM for MMP-7,
2.7 nM for MMP-9, and 2.15 nM for MMP-14) and
the inhibitor solutions were incubated in the assay buffer
for 4 h at 25 °C. After addition of 200 lM solution of
the fluorogenic substrate, Mca-Pro-Leu-Gly-Leu-Dpa-
Ala-Arg-NH2 (Sigma), in DMSO (final concentration
2 lM), the hydrolysis was monitored every 15 s, for
20 min, recording the increase in fluorescence
(kex = 328 nm, kem = 398 nm) using a Molecular Device
M-2 Gemini plate reader. The assays were performed
in duplicate in a total volume of 200 ll per well in 96-
well microtiter plates (Corning, black, NBS). Control
wells lack an inhibitor. The MMP inhibition activity
was expressed in relative fluorescent units (RFU). Per-
cent of inhibition was calculated from control reactions
without the inhibitor. IC50 was determined using the for-
mula: Vi/Vo = 1/(1 + [I]/IC50), where Vi is the initial
velocity of substrate cleavage in the presence of the
inhibitor at concentration [I] and Vo is the initial veloc-
ity in the absence of the inhibitor. Results were analyzed
using SoftMax Pro software and GraFit software.52,53
5.3. Cell culture studies
The lung carcinoma cell line A549 was obtained from
American Type Culture Collection. The prostate carci-
noma cell lines Tsu-Pr1 and PPC-1 were a kind gift from
Dr. James Norris, Medical University of South Caroli-
na. Cells were maintained in RPMI-1640 supplemented
with 10% heat-inactivated fetal bovine serum, 2 mM
glutamine, and 1 mM sodium pyruvate (complete medi-
um). In experiments with depleted folate supplementa-
tion, cells were grown on folate-free media
supplemented with 10% dialyzed PBS and 20 nM leuco-
vorin. All cells were grown at 37 °C under humidified air
containing 5% CO2. Cells were plated in 96-well plates at
a density of about 10,000 cells/well. Treatment with dif-
ferent concentrations of a corresponding inhibitor was
performed constantly for 72 h. MTT cell proliferation
assay was performed using CellTiter 96 kit (Promega)
according to manufacturer’s directions.
5.3.1. Assays of DHFR activity. DHFR activity, in the
presence and in the absence of inhibitors, has been as-
sayed spectrophotometrically by monitoring the de-
crease in absorbance at 340 nm due to oxidation of
NADPH to NADP+. The enzyme, purified from
MTX-resistant L. casei strain, was a kind gift from
Dr. Priest. L. casei DHFR has a fold similar to the hu-
man enzyme, possesses the conserved residues important
for MTX binding, and it has been used in a number of
studies as a surrogate of human DHFR demonstrating
a similar inhibitory pattern for MTX and its ana-
logs.46–50 All assays were performed at 30 °C in a Shi-
madzu 2401PC double-beam spectrophotometer. The
reaction mixture contained 100 lM of folic acid or
25 lM of dihydrofolic acid, 150 lM NADPH, and
1.0–2.5 lg/mL (0.006–0.015 U/ml) DHFR. The reaction
was started by the addition of the enzyme in a final vol-
ume of 1.0 ml and read against a blank cuvette contain-
ing all components except the enzyme. MTX-derivatives
were added in the reaction mixture to final concentra-
tion 0.001–1.0 lM. Derivatives of folic acid were tested
in the concentration range of 0.1–100 lM.
Acknowledgments
This work was supported by the Portuguese Fundac¸a˜o
ˆ
para a Ciencia e Tecnologia (SFRH/BPD/11653/2002,
MAS) and by the National Institute of Health Grant
DK54388 (S.A.K.).
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