Novel folate-hydroxamate based antimetabolites: Synthesis and biological evaluation

Article abstract of DOI:10.2174/157340611796150923

A set of hydroxamate derivatives of folic acid and methotrexate (MTX) was synthesized and evaluated for the inhibitory activity against histone deacetylase (HDAC) and dihydrofolate reductase (DHFR), two enzymes overexpressed in metastasizing tumors. The s

Full text of DOI:10.2174/157340611796150923

Medicinal Chemistry, 2011, 7, 265-274
265
Novel Folate-Hydroxamate Based Antimetabolites: Synthesis and Bio-
logical Evaluation
Marta P. Carrascoa, Eva A. Enyedya, Natalia I. Krupenkob, Sergey A. Krupenkob, Elisa Nutic,
Tiziano Tuccinardic, Salvatore Santamariac, Armando Rosselloc, Adriano Martinellic and M. Amélia
Santosa*
aCentro de Química Estrutural, Instituto Superior Técnico, 1049-001 Lisboa, Portugal
bDepartment of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, 29425,
USA
cDipartimento di Scienze Farmaceutiche, Università di Pisa, Via Bonanno 6, 56126, Pisa, Italy
Abstract: A set of hydroxamate derivatives of folic acid and methotrexate (MTX) was synthesized and evaluated for the
inhibitory activity against histone deacetylase (HDAC) and dihydrofolate reductase (DHFR), two enzymes overexpressed
in metastasizing tumors. The synthesized compounds were further screened for their antiproliferative activity in two hu-
man cancer cell lines, A549 (non-small cell lung carcinoma) and PC-3 (prostate adenocarcinoma). All derivatives showed
significant inhibitory activity against HDACs (micromolar range) while only the MTX derivative was reasonably effec-
tive in DHFR inhibition. A docking study provided insight into the binding mode of the most potent inhibitor in the active
sites of the enzymes, allowing rationalization of the bioassays. The MTX-based compound could be of interest for testing
against metastasizing tumors in an animal model. The studied derivatives represent promising molecular templates for fur-
ther development of dual activity anti-cancer drugs.
Graphical Abstract:
COOH
O
HN
O
HN
R1
N
O
Spacer
NHOH
N
N
N
N
R2
H2N
DHFR
HDAC
Keywords: Antiproliferative, cancer therapy, dihydrofolate reductase inhibitors, dual enzyme inhibitors, folates and antifolates,
histone deacetylase inhibitors, methotrexate.
INTRODUCTION
longs to a class of drugs known as antifolates. It inhibits di-
hydrofolate reductase (DHFR), a key enzyme in folate me-
tabolism [5,6], by binding strongly to the hydrophobic fo-
late-binding pocket of the enzyme [7]. The downstream
mechanism, associated with targeting of DHFR by MTX, is
the inhibition of the de novo synthesis of dTMP and purine
nucleotides with ultimate blockage of DNA biosynthesis.
This further results in the accumulation of DNA damage and
cytotoxicity [7,8]. Of note, MTX has been successfully used
in combination with other drugs for treatment of cancer [5].
One of the promising recent strategies for cancer therapy
is based on the generation of compounds affecting multiple,
sometimes unrelated, cancer cell targets [1-3]. Methotrexate
(MTX), one of the oldest anticancer drugs in clinical use [4],
is an attractive candidate to be used as a carrier for the sec-
ond chemical pharmacophore in this hybrid drug approach.
Although MTX is a structural analogue of folic acid, it be-
Another class of rapidly emerging chemotherapeutic
agents is histone deacetylase (HDAC) inhibitors (HDACIs)
[9]. HDACs are important regulators of chromatin remodel-
*Address correspondence to this author at the Centro de Química Estrutural,
Instituto Superior Técnico, 1049-001 Lisboa, Portugal; Tel: +351 21 841 92
73; Fax: +351 21 846 44 55; E-mail: masantos@ist.utl.pt
1573-4064/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

266 Medicinal Chemistry, 2011, Vol. 7, No. 4
Carrasco et al.
NHOH
COOH
O
1: R=
3: R=
O
R
O
HN
HN
O
OH
N
NHOH
O
N
N
H
H2N
N
N
4: R=
COOH
O
HN
O
HN
HN
NH2
NHOH
N
O
N
N
H2N
N
N
O
NHOH
O
OH
N
H
O
H2N
Fig. (1). Structures of the synthesized compounds.
ing and their inhibition drastically alters gene expression and
induces apoptosis in tumor cells [10]. In addition to histones,
HDACs target other acetylated proteins, including a variety
of transcription factors [11]. Therefore, the antiproliferative
effects of HDACIs likely involve multiple mechanisms. The
inhibitors can change the expression of 7-10% of genes and
induce differentiation, cell-cycle arrest, and apoptosis in
transformed cell cultures [12,13]. In October 2006, the US
FDA approved one such inhibitor, SAHA, for the treatment
of cutaneous T-cell lymphoma [14], and several new inhibi-
tors are in different phases of clinical trials at present [9].
to gain insight into the molecular interactions responsible for
the observed biological effects.
RESULTS AND DISCUSSION
Molecular Design
The strategy for the design of the dual-targeting com-
pounds is based on a combination, in a single molecule, of
the two active functional groups capable of targeting and
inhibiting two different enzymes. Specifically, the newly
synthesized compounds include the aminopteridine nucleus,
to enable the DHFR inhibition, and a primary hydroxamate
moiety, as the zinc-binding group (ZBG), to inhibit HDAC
via coordination to its zinc-cofactor. The two moieties are
connected through long flexible spacers (similarly to SAHA)
to enable the ZBG access to the zinc ion bound in the HDAC
active site (this ion is situated on the bottom of a long narrow
channel in the protein molecule). To preserve the structural
determinants of MTX, essential for the binding with DHFR,
the molecule was modified by attaching the aminocaproic
hydroxamic acid to the ꢀ-carboxylic group of the glutamate.
The corresponding folic acid derivatives were also synthe-
sized and studied. The rationale for studying these deriva-
tives was a potential for increased delivery of hydroxamate
due to the elevated levels of folate receptor in cancer cells
[23]. Since the potency of the HDAC inhibitors depends on
their accommodation at the active site of the enzyme, the
folic acid derivatives were designed with different spacer
groups between the core molecule and the hydroxamate
ZBG, namely through variation of the chain length and inser-
tion of phenyl group para-aminobenzoic acid (pABA)-
caproic hydroxamic acid was also synthesized as the simple
analogue model, containing only one of the two functional
groups. Overall, the following derivatives of MTX and folic
acid were synthesized and tested (Fig. 1): folate-caproic hy-
droxamic acid (1), methotrexate-caproic hydroxamic acid
The recent paradigm of multifunctional pharmacology
emerged as a rather efficient way to treat complex diseases,
such as cancer. It involves the use of either multiple drugs
directed toward multiple targets or the use of a single com-
pound with multiple activities [15]. Recent studies reported
the use of HDACIs in combination with other anticancer
drugs, namely with 5-fluorouracil, an antimetabolite used to
treat breast and colorectal cancers [16]. The enhanced effi-
cacy of these inhibitor combinations suggests very promising
future therapeutic applications of HDACIs [17-19].
Following our recent research interest in the development
of multifunctional compounds for the treatment of complex
multifactorial diseases [20], in particular, the use of anti-
folates bearing an additional functional group [21,22], we
have developed a series of hybrid compounds for simultane-
ous inhibition of DHFR and HDACs, two important targets
in drug development for the treatment of several diseases.
These compounds are based on the combination of two main
functional groups, MTX/folate and hydroxamate which are
connected by different spacers to allow for optimization of
the ligand-enzyme interactions. Herein we describe the de-
sign and synthesis of the compounds and their inhibitory
activities against DHFR and HDAC8, and their anti-
proliferactive effects on two human cancer cell lines, A549
and PC3. We also employed a molecular docking approach

Novel Folate-Hydroxamate Based Antimetabolites
Medicinal Chemistry, 2011, Vol. 7, No. 4 267
COOH
COOH
O
O
HN
HN
i, ii/i, iii
OH
HN
R1
O
R1
N
O
R3
N
N
N
N
N
N
N
N
R2
R2
H2N
N
H2N
Follic acide
MTX
R1=OH; R2=H; R=(CH2)5CONHOH
R1=NH2; R2=CH3; R3= (CH2)5CONHOH
R1=OH; R2=H2; R3= PhCONHOH
1
2
3
R1=OH; R2=H
R1=NH2; R2=CH3
COOH
O
O
HN
COOH
O
HN
OH
N
O
HN
N
N
HN
H
OH
N
i, ii/i, iii
4
H2N
N
N
O
N
N
H
O
NHOH
NHOH
H2N
N
N
9
O
O
O
OH
OH
OH
N
H
iv
i, ii
Boc
Boc
O
H2N
N
N
H
H
pABA
10
11
i, iii
O
O
NHOH
NHOH
N
v
N
H
H
Boc
O
O
N
12
H2N
H
5
Scheme 1. Synthesis of the ꢀ-hydroxamic acid derivatives of MTX and folic acid and pABA-caproic hydroxamic acid. Reagents and condi-
o
o
o
tions: (i) TBTU, NMM, 0 C, dry DMF; (ii) aminoacid (6-amino-caproic acid, pABA), 0 C, dry DMF; (iii) NH2OH, 0 C, dry DMF; (iv)
Na2CO3, Boc2O, 0 oC, water, 1,4-dioxane; (v) TFA, CH2Cl2, RT.
This last step involves, once more, the use of TBTU as the
activating agent.
(2), folate-pABA hydroxamic acid (3), folate-pABA-caproic
hydroxamic acid (4) and pABA-caproic hydroxamic acid (5).
With regard to pABA-caproic hydroxamic acid (5), the
preparation of the corresponding precursor, pABA-caproic
acid, involved an initial protection of the pABA amino group
with Boc, with standard deprotection using TFA as the last
step.
Chemistry
The methotrexate derivative 2 and the folic acid deriva-
tives 1, 3, and 4 were synthesized as described in Scheme 1.
The first step of the synthetic process involved the cou-
pling of an amino group of amino acid molecules (6-amino-
caproic acid or pABA) to the ꢀ-carboxylic group of folic acid
or MTX, with the preceeding carboxylic group activation by
stoichiometric amount of TBTU in the presence of N-
methyl-morpholine (NMM). The rationale for this carboxylic
activation (use of a bulky activating group and strict equiva-
lent amount of folate/MTX and TBTU) was the promotion of
the coupling of the amine group to the ꢀ-carboxylic group
due to hindrance of the ꢀ-position by the bulky activating
agent. For the precursor of folate-pABA-caproic hydroxamic
acid (5), the 6-amino-caproic acid was coupled to the acti-
vated folate-pABA acid. The last step was always the con-
version of the terminal carboxylic group of the new amino
acid derivative to the corresponding hydroxamic acid ana-
logue through condensation with the free hydroxylamine.
HDACs Inhibition
The synthesized derivatives and the reference compound
Trichostatin A (TSA) were tested for HDAC inhibition. Re-
combinant human HDAC8 or crude nuclear extract from
HeLa cells were used as target enzymes in fluorimetric as-
says (Table 1). HDAC8 isoform was selected for the assays
since its X-ray crystal structure is currently available. This
enabled us to correlate the inhibitory activity and the interac-
tions between the inhibitors and targeted active sites.
With regard to HDAC8 inhibition, among all derivatives,
the model pABA-caproic hydroxamic acid (5) showed quite
good inhibitory activity with IC50 value in a micromolar
range, lower than those for the aminocaproic acid derivatives
of folate or MTX. Folate-caproic hydroxamic acid (1) and

268 Medicinal Chemistry, 2011, Vol. 7, No. 4
Carrasco et al.
Table 1. Inhibitory Activities of the ꢀ-hydroxamic Acid Derivatives of MTX and Folate and of the pABA-caproic Hydroxamic Acid,
as well as the Reference Drug (TSA) Against Human HDAC8 and the HDACs of the Crude Nuclear Extract from HeLa
Cells (IC50, μM)
Compound
Human HDAC8
HeLa Nuclear Extract
folate-caproic hydroxamic acid (1)
methotrexate-caproic hydroxamic acid (2)
folate-pABA hydroxamic acid (3)
folate-pABA-caproic hydroxamic acid (4)
pABA-caproic hydroxamic acid (5)
TSA
23±4
29±5
5.5±0.3
5.7±0.2
1.9±0.1
0.88±0.08
0.34±0.01
0.005
10.8±1
6.6±0.8
13±1.7
1
methotrexate-caproic hydroxamic acid (2) demonstrated
similar inhibitory activities, due to obvious structural simi-
larities. Probably only minor differences in additional secon-
dary interactions within the HDAC catalytic site took place.
Changing the linker chain from the amino-caproic acid
(compound 1) to the pABA (compound 3) leads to an in-
creased inhibitory activity (IC50 decreased from 23 to 10.8
small cell lung carcinoma) and PC-3 (human prostate cancer)
cells (Table 2). The model compound 5 presented a low IC50
value, which could not be rationalized by the direct interac-
tion with the active site of DHFR, but rather by the effect on
other important enzymes overexpressed in cancer cells,
namely HDAC (due to the demonstrated above high inhibi-
tory activity of this compound against the enzyme). Among
the synthesized compounds, the MTX derivative presented
much stronger anti-proliferative activity than the folate de-
rivatives. Nevertheless, its activity was considerably weaker
than that of MTX itself, which could be explained by the
lack of intracellular polyglutamylation due to replacement of
the MTX ꢀ-carboxylic group by the hydroxamate moiety.
Since folic acid is not a DHFR inhibitor, but one of its sub-
strates, and it is not toxic even at high concentrations, the
lack of activity for the folic acid derivatives was somewhat
anticipated. The folic acid moiety was expected to enhance
the transport of the attached hydroxamate into the cells, in-
crease the effective concentration of HDACI and suppress
proliferation through folate-independent pathways. Appar-
ently, this was not the case in our experiments: IC50 for these
compounds in both cell lines were over 100 times higher
than their respective IC50 towards the HeLa nuclear HDACs.
Also, none of the tested inhibitors was as effective as MTX
itself (IC50 for MTX is 1 nM, while for all derivatives it is in
medium-high micromolar range). In general, among the
studied compounds, the MTX hydroxamate demonstrated the
best inhibitory activity against DHFR (micromolar range).
This finding suggests that the growth suppressor effects re-
sulted from DHFR inhibition and these should be attributed
to the MTX component.
μM). The most active compound of this series was 4 (IC50
=
6.6 μM), the one with the longest linker chain (a conjugation
of pABA with amino-caproic acid). These results suggest
correlation between the spacer length and the inhibitory ef-
fect. Apparently, this phenomenon may be associated with
greater accessibility of the HDAC-bound zinc to the hydrox-
amate more remotely positioned from the bulky pteridine
core. Similarly, introduction of such a linker into MTX de-
rivative might increase the interactions with the active site of
HDAC, and improve the inhibitor’s activity.
All compounds were also tested for their inhibitory activ-
ity against a mix of HDACs, using the crude nuclear extract
from HeLa cells (human cervical cancer cell line) as the
source of HDACs (Table 1). This assay provided IC50 values
5-fold lower than those obtained for isolated HDAC8. Com-
pound 4 was still the most active folate derivative, with IC50
of 0.88 μM. Surprisingly, compound 5 showed a higher ac-
tivity against mixed HDACs (IC50 = 0.34 μM). The refer-
ence compound TSA was also more active in this assay than
with HDAC8.
DHFR Inhibition
Inhibitory activities of the synthesized MTX and folic
acid derivatives against DHFR are summarized in Table 2.
Purified L. casei DHFR was used for evaluation of inhibitory
activities of the compounds. L. casei enzyme proved to be a
satisfactory model for such experiments previously [24].
With no surprise, our experiments revealed that the MTX
derivative 2 displayed the strongest activity toward DHFR
with the IC50 value in a hundreds nanomolar range. In con-
trast, the folate analogs showed weak inhibition (high mi-
cromolar range). Compared to MTX, however, the hydrox-
amate derivative of MTX displayed considerably lower
DHFR inhibitory activity.
Docking Studies
To gain an idea of the structural basis for the observed
inhibitory effects we have evaluated the binding characteris-
tics of the active molecules using docking studies of folate-
pABA-caproic hydroxamic acid (4) and methotrexate-
caproic hydroxamic acid (2), in the active site of HDAC8
and DHFR, respectively. The program, GOLD, was used for
docking calculations after an extensive conformational
search using the available scoring functions (GoldScore,
ChemScore and ASP). Application of the GoldScore func-
tion by the GOLD program allowed successful prediction of
the arrangements of the ligands co-crystallized in the respec-
tive enzyme active site: SAHA for HDAC8 and MTX in the
Cell Proliferation Studies
The antiproliferative activities of the hydroxamate de-
rivatives of folate and MTX were evaluated on A549 (non-

Novel Folate-Hydroxamate Based Antimetabolites
Medicinal Chemistry, 2011, Vol. 7, No. 4 269
Table 2. Inhibitory Activities of the Synthesized Compounds and Reference Drug MTX Against L. casei DHFR and Antiproliferative
Effect on Cancer Cell Liynes (IC50, μM)
Antiproliferative Activities
Compound
L. casei DHFR
A594 Cells
PC-3 Cells
(ꢀ10)
(ꢀ10)
folate-caproic hydroxamic acid (1)
methotrexate-caproic hydroxamic acid (2)
folate-pABA hydroxamic acid (3)
folate-pABA-caproic hydroxamic acid (4)
pABA-caproic hydroxamic acid (5)
MTX
60
0.25
360
20
45
5
70
6
46
70
70
70
650
0.005
9
50
0.0001
0.0001
Fig. (2). Molecular docking of folate-pABA-caproic hydroxamic acid (4) into the active-site pocket of human recombinant HDAC8 (left)
and methotrexate-caproic hydroxamic acid (2) into Lactobacillus casei DHFR (rigth).
pteridine amine group and Asp101 (2.07 Å). Due to the con-
formational arrangement in this inhibitor, it interacts in a
suitable manner with the hydrophobic residues of the outside
surface of the protein. The presence of this ꢀ-ꢀ interaction
between a phenyl moiety of the inhibitor and the Tyr100
residue, contributes to increase the favorable interactions
with the enzyme’s surface. The additional hydrogen bond
contributes to the enhanced binding of the pteridine ring,
which otherwise is retained by non-hydrophobic interactions
with residues on the outer surface of the enzyme.
case of DHFR. Therefore, the same procedure was used for
the docking of the synthesized ligands inside the active site
of the two enzymes.
In order to gain insight into the binding mode of the most
potent inhibitor in HDAC8 active site, a docking approach
was employed using as a model the catalytic core of human
HDAC8 (Fig. 2. left). This inhibitor (4) is able to accommo-
date its long aliphatic chain into the enzyme’s pocket and
make several contacts with the tube-link hydrophobic por-
tion of the pocket establishing good interactions. The termi-
nal hydroxamic acid group reaches the polar bottom of the
pocket where it coordinates the zinc ion in a bidentate fash-
ion while interacting simultaneously with His142 and
His143, residues likely to be involved the catalytic process
[25, 26]. In the ligand chain connecting the linker and the
cap group, the amide function establishes a strong hydrogen
bond with Asp101 (1.99 Å). The aromatic cap group of this
ligand consists of a phenyl-amino group that interacts with
the Tyr100 residue by a hydrogen bond (2.04 Å). An addi-
tional significant hydrogen bond is formed between the
The same approach was used to assess the binding mode
of the most potent derivative (2) into DHFR active site (Fig.
2. rigth). For this docking calculation, a model of the cata-
lytic core of L. casei DHFR was used. Inhibitors of DHFR
usually include a 2,4-diamino substitution in the pteridine
ring which binds to DHFR active site establishing important
hydrogen bonds with the Asp26 (1.88 Å), Ala97 (2.40 Å)
and Leu4 (1.55 Å) amino acid residues. Some significant
interactions can also be observed between the pteridine ring
of the MTX derivative and the hydrophobic portion of the

270 Medicinal Chemistry, 2011, Vol. 7, No. 4
Carrasco et al.
enzyme active site. Furthermore, the presence of ꢀ-ꢀ interac-
tion between this planar moiety and Phe30 should give an
important contribution to the good inhibition values found
for this derivative. Additional hydrogen bond interactions
can be found between the carboxylic oxygen atoms present
in the flexible moiety of this inhibitor and the amino acid
residues Arg57 and His28. In conclusion, all these favorable
interactions between the inhibitor and the aminoacid residues
contribute to enclose it successfully into the active site and,
consequently, obtain satisfactory inhibition values.
from sodium 3-(trimethylsilyl)-[2,2,3,3-2H4]-propionate as
internal reference in D2O solutions. The following abbrevia-
tions are used: d = doublet; s = singlet; t = triplet; m = mul-
tiplet; br = broad. Mass spectra (FAB) were performed on a
VG TRIO-2000 GCMS instrument. High Resolution Mass
Spectrometry (HRMS (ESI)) measurements were performed
on an APEX III FT-ICR MS (Bruker Daltonics, Billerica,
MA), equipped with a 7T actively shielded magnet. Ions
were generated using an Apollo API electrospray ionization
(ESI) source. Ionization was achieved by an electrospray
ionization source (Bruker Daltonics, Billerica, MA), with a
voltage of 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
CONCLUSIONS
A set of novel hybrid compounds, namely hydroxamate
derivatives of methotrexate (MTX) and folic acid, have been
developed and evaluated for their ability to inhibit HDAC8
and DHFR, two enzymes with important roles in tumor cells.
The anti-proliferativite activity of the compounds in two
cancer cell lines (lung carcinoma and human prostate adeno-
carcinoma) was further evaluated.
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 to 5% to give the best signal-to-noise ratio. Data
acquisition and data processing were performed using the
XMASS software, version 6.1.2 (Bruker Daltonics). Elemen-
tal analyses were performed on a Fisons EA 1108 CHNF/O
instrument.
The folate and MTX derivatives showed a reasonably
good ability to inhibit both HDAC and DHFR. With regard
to HDAC inhibition, folate-pABA-caproic hydroxamic acid
(4) was the most active; despite its large size, it can form
strong interactions with the enzyme catalytic site. This inhi-
bition is quite independent of the pteridine-ring 4-substituent
(amino for MTX; hydroxyl for folic acid). Concerning the
DHFR inhibition and the antiproliferative effects on lung
carcinoma cells, the MTX derivative 2 was the most active
compound, as expected. Thus, it can be hypothesized that if
some of the derivatizations made on folic acid (e.g. com-
pound 3 and 4) were extended to MTX, it would result on
MTX derivatives with high inhibitory activity against both
DHFR and HDAC8. The fact that the studied derivatives
retain the capability to inhibit two independent targets pro-
vides directions for design strategies of novel hybrid com-
pounds with improved dual activity against HDACs and
DHFR and for improved treatment of cancer including me-
tastasizing tumors.
Synthesis
(S)-6-(4-(4-(((2-Amino-4-hydroxypteridin-6-yl)methyl)
amino)benzamido)-4-carboxybutanamido)hexanoic
(Folate-caproic acid, 6).
acid
A first solution mixture was prepared by adding N-
methylmorpholine (0.048 mL, 0.44 mmol) to a solution of
folic acid (0.100 g, 0.22 mmol) in dry DMF (10 mL) under
º
nitrogen and this solution was cooled in an ice bath at 0 C
followed by the addition of TBTU (0.071 g, 0.22 mmol).
This solution was slowly added to a water-ice cooled solu-
tion of 6-amino caproic acid (0.036 g, 0.22 mmol) in dry
DMF (10 mL) and the reaction mixture was stirred for 2 h at
0 ºC and then it was left to heat up to RT and left stirring for
12 h. DMF was evaporated under high vacuum. Recrystalli-
zation of the solid residue from a mixture of dry di-
chromethane/ethanol provided the pure product as yellow
1
crystals (67%); H NMR (D2O, pD = ca. 9) ꢁ: 8.59 (1H, s,
CH=N), 6.80 (2H, d, CH=C–NH), 7.65 d (2H, d, CH=C–
C=O), 4.57 (2H, s, CH2–NH), 4.34 (1H, s, CH–COOH),
2.91 (2H, t, NH-CH2-CH2–CH2–CH2-CH2), 2.31 (2H, t,
CH2–C=O), 2.14 (2H, t, NH-CH2-CH2–CH2–CH2-CH2), 2.04
(2H, d, CH2–CH), 1.45 (4H, m, NH-CH2-CH2–CH2–CH2-
CH2, NH-CH2-CH2–CH2–CH2-CH2), 1.18 (2H, q, NH-CH2-
CH2–CH2–CH2-CH2); IR: 1541 cm-1 (ꢂNH(CONH), new amide
bond); MS (FAB) m/z: 555 (50%) (M+1).
EXPERIMENTAL
Chemicals and Equipment
All the commercially available reagents were of the
highest purity and were used without further purifications.
Folic acid, methotrexate, O-benzotriazol-1-yl-N,N,N’,N’-
tetramethyluronium-tetrafluoroborate
(TBTU),
N-
methylmorpholine (redistilled), NH2OH, di-tert-butyl dicar-
bonate were purchased from Sigma-Aldrich and 6-amino-
caproic acid, 4-amino-benzoic acid from Acros Organics.
The solvents were purchased from Acros Organics or Merck
and, whenever necessary, they were purified and dried ac-
cording to standards methods [27]. All moisture-sensitive
reactions were performed under nitrogen atmosphere. The
chemical reactions were followed by TLC using silica gel
plates (G-60 F254, Merck). A Bio-Rad Merlin, FTS 3000 MX
spectrometer was used to record solid state IR spectra (KBr
pellets). 1H and 13C NMR spectra were recorded on a Varian
(S)-2-(4-(((2-Amino-4-hydroxypteridin-6-yl)methyl)
amino)benzamido)-5-((6-(hydroxyamino)-6-oxohexyl)amino)
-5-oxopentanoic acid (Folate-caproic hydroxamic acid, 1).
A cooled solution mixture containing the activated folate-
caproic acid was slowly added to a water-ice cooled solution
of the free hydroxylamine prepared under nitrogen, by add-
ing potassium hydroxide as pellets (0.05 g, 0.85 mmol) to a
solution of hydroxylamine hydrochloride (0.06 g, 0.85
mmol) in dry DMF (10 mL), containing activated molecular
º
sieves (0.4 nm, Riedel-de Haën), in an ice bath at 0 C. The
º
Unity 300 FT NMR spectrometer at 25 C. If necessary, as-
mixture was stirred for 30 min and the inorganic solids were
filtered out from the solution. The first solution was added
slowly to the second one and the mixture was left stirring for
signment of the signals of the 13C NMR signals were con-
firmed by DEPT. Chemical shifts are reported in ppm (ꢁ)

Novel Folate-Hydroxamate Based Antimetabolites
Medicinal Chemistry, 2011, Vol. 7, No. 4 271
º
5 h at 0 C and the solution was kept at this temperature for
COOH), 41.7 (NH–CH2–CH2–CH2–CH2–CH2), 39.9 (CH3),
35.2 (NH–CH2–CH2–CH2–CH2–CH2), 33.2 (CH2–C=O),
30.2 (NH–CH2–CH2–CH2–CH2–CH2), 28.5 (NH–CH2–CH2–
CH2–CH2–CH2), 27.9 (NH–CH2–CH2–CH2–CH2–CH2), 27.4
(CH2–CH); IR: 1644 cm-1 (ꢁC=O, new amide bond). Anal.
found: C, 53.49; H, 5.92; N, 24.23%; calculated for
C26H34N10O6: C, 53.60; H, 6.03; N, 24.04%.
12 h. After evaporation of the solvent (DMF) under high
vacuum, the solid residue was taken into CH2Cl2 and by the
addition of dry ethanol yellow solids were precipitated, fil-
tered and washed with ethanol and diethyl ether in the end.
Recrystallization of these solid from DMF provided yellow
crystals as pure product (83%); m.p. 250-260 ºC (decomposi-
tion). 1H NMR (D2O, pD ca. 9) ꢀ: 8.62 (1H, s, CH=N), 7.68
d (2H, d, CH=C–C=O), 6.86 (2H, d, CH=C–NH), 4.62 (2H,
s, CH2–NH), 4.33 (1H, s, CH–COOH), 2.97 (2H, t, NH-
CH2-CH2–CH2–CH2-CH2), 2.33 (2H, t, CH2–C=O), 2.15
(2H, t, NH-CH2-CH2–CH2–CH2-CH2), 2.06 (2H, d, CH2–
CH), 1.54 (4H, m, NH-CH2-CH2–CH2–CH2-CH2, NH-CH2-
CH2–CH2–CH2-CH2), 1.29 (2H, q, NH-CH2-CH2–CH2–CH2-
CH2); 13C NMR (DEPT) (D2O, pD = ca. 9) ꢀ: 150 (CH=N),
132 (CH=C–C=O), 115 (CH=C–NH), 57.5 (CH–COOH),
48.2 (CH2-NH), 41.8 (NH–CH2–CH2–CH2–CH2–CH2) , 39.9
(NH–CH2–CH2–CH2–CH2–CH2), 35.0 (CH2–C=O), 30.3
(CH2–CH), 28.4 (NH–CH2–CH2–CH2–CH2–CH2), 27.9
(NH–CH2–CH2–CH2–CH2–CH2), 27.3 (NH–CH2–CH2–
CH2–CH2–CH2); IR: 1614 cm-1 (ꢁC=O, new amide bond) 1547
cm-1, 1521 cm-1 (ꢁNH(CONH), new amide bond). Anal. found:
C, 52.92; H, 5.63; N, 22.20%; calculated for C25H31N9O7: C,
52.72; H, 5.49; N, 22.13%.
(S)-4-(4-(4-(((2-Amino-4-hydroxypteridin-6-yl)methyl)
amino)benzamido)-4-carboxybutanamido) benzoic acid (Fo-
late-pABA, 8).
From folic acid and pABA, as in the case of folate-
1
caporic acid. Yellow crystals (62%); H NMR (D2O, pD ca.
9) ꢀ: 8.51 (1H, s, CH=N), 7.65 d (2H, d, CH=C–C=O), 7.44
(2H, d, NH-C=CH(pABA)), 7.25 (2H, d, CH=C-
CO(pABA)), 6.63 (2H, d, CH=C–NH), 4.49 (2H, s, CH2–
NH), 4.42 (1H, s, CH–COOH), 2.36 (2H, t, CH2–C=O), 2.31
(2H, d, CH2–CH); 13C NMR (DEPT) (D2O, pD = ca. 9) ꢀ:
150 (CH=N), 132 (CH=C-CO(pABA)), 131 (CH=C–C=O),
124 (NH-C=CH(pABA)), 115 (CH=C–NH), 57.7 (CH–
COOH), 48.4 (CH2-NH), 36.2 (CH2–C=O), 30.2 (CH2–CH);
IR: 1654 cm-1 (ꢁC=O, new amide bond), 1540 cm-1, 1510 cm-1
(ꢁNH(CONH), new amide bond); MS (FAB) m/z: 561 (85%)
(M+1).
(S)-2-(4-(((2-Amino-4-hydroxypteridin-6-yl)methyl)
amino)benzamido)-5-((4-(hydroxycarbamoyl)phenyl)amino)-
5-oxopentanoic acid (Folate-pABA hydroxamic acid, 3).
(S)-6-(4-Carboxy-4-(4-(((2,4-diaminopteridin-6-yl)
methyl)(methyl)amino)benzamido)butanamido)
hexanoic
acid (Methotrexate-caproic acid, 7).
From folate-pABA, as in the case of folate-caproic hy-
From MTX and 6-amino-caproic acid, as in the case of
folate-caproic acid. Yellow crystals (24%); H NMR (D2O,
1
droxamic acid. Yellow crystals (92%); m.p. 210-220 °C (de-
1
composition). H NMR (D2O, pD = ca. 9) ꢀ: 8.53 (1H, s,
pD = ca. 9) ꢀ: 8.55 (1H, s, CH=N), 7.65 d (2H, d, CH=C–
C=O), 6.84 (2H, d, CH=C–NH), 4.80 (2H, s, CH2–NCH3),
4.37 (1H, s, CH–COOH), 3.16 (3H, s, CH3), 2.84 (2H, t,
NH-CH2-CH2–CH2–CH2-CH2), 2.30 (2H, t, CH2–C=O), 2.10
(2H, t, NH-CH2-CH2–CH2–CH2-CH2), 1.98 (2H, d, CH2–
CH), 1.49 (4H, m, NH-CH2-CH2–CH2–CH2-CH2, NH-CH2-
CH2–CH2–CH2-CH2), 1.17 (2H, q, NH-CH2-CH2–CH2–CH2-
CH2); 13C NMR (DEPT) (DMSO-d6) ꢀ: 147 (CH=N), 129
(CH=C–C=O), 111 (CH=C–NH), 54.6 (CH–COOH), 52.8
(CH2-NCH3), 32.1 (CH2–C=O), 28.9 (NH–CH2–CH2–CH2–
CH2–CH2), 27.2 (CH2–CH), 26.0 (NH–CH2–CH2–CH2–
CH2–CH2), 24.3 (NH–CH2–CH2–CH2–CH2–CH2), covered
by the solvent peak (CH3, NH–CH2–CH2–CH2–CH2–CH2,
NH–CH2–CH2–CH2–CH2–CH2); IR: 1646 cm-1 (ꢁC=O, new
amide bond); MS (FAB) m/z: 568 (100%) (M+1).
CH=N), 7.64 d (2H, d, CH=C–C=O), 7.49 (2H, d, NH-
C=CH(pABA)), 7.30 (2H, d, CH=C-CO(pABA)), 6.68 (2H,
d, CH=C–NH), 4.52 (2H, s, CH2–NH), 4.46 (1H, s, CH–
COOH), 2.17 (2H, t, CH2–C=O), 2.11 (2H, d, CH2–CH); 13
C
NMR (DEPT) (D2O, pD = ca. 9) ꢀ: 150 (CH=N), 132
(CH=C-CO(pABA)), 131 (CH=C–C=O), 123 (NH-
C=CH(pABA)), 115 (CH=C–NH), 57.9 (CH–COOH), 48.4
(CH2-NH), 33.9 (CH2–C=O), 30.1 (CH2–CH); IR: 1658 cm-1
(ꢁC=O, new amide bond), 1539 cm-1, 1514 cm-1 (ꢁNH(CONH)
,
new amide bond). Anal. found: C, 53.41; H, 4.59.; N,
21.76%; calculated for C26H25N9O7: C, 53.26; H, 4.38; N,
21.90%.
(S)-6-(4-(4-(4-(((2-Amino-4-hydroxypteridin-6-yl)methyl)
amino)benzamido)-4-carboxybutanamido) benzamido)hex-
anoic acid (Folate-pABA-caproic acid, 9).
(S)-2-(4-(((2,4-Diaminopteridin-6-yl)methyl)(methyl)
amino)benzamido)-5-((6-(hydroxyamino)-6-oxohexyl)amino)
-5-oxopentanoic acid (Methotrexate-caproic hydroxamic
acid, 2).
From folate-pABA and 6-amino-caproic acid, as in the
1
case of folate-caproic acid. Yellow crystals (66%); H NMR
(D2O, pD = ca. 9) ꢀ: 8.56 (1H, s, CH=N), 7.84 (2H, d,
CH=C-CO(pABA)), 7.67 (2H, d, NH-C=CH(pABA)), 7.51
d (2H, d, CH=C–C=O), 6.78 (2H, d, CH=C–NH), 4.58 (2H,
s, CH2–NH), 4.38 (1H, s, CH–COOH), 3.00 (2H, t, NH-
CH2-CH2–CH2–CH2-CH2), 2.40 (2H, t, CH2–C=O), 2.12
(4H, m, CH2–CH, NH-CH2-CH2–CH2–CH2-CH2), 1.48 (6H,
br, NH-CH2-CH2–CH2–CH2-CH2, NH-CH2-CH2–CH2–CH2-
CH2, NH-CH2-CH2–CH2–CH2-CH2); 13C NMR (DEPT)
(D2O, pD = ca. 9) ꢀ: 150 (CH=N), 132 (CH=C-CO(pABA)),
131 (CH=C–C=O), 123 (NH-C=CH(pABA)), 114 (CH=C–
NH), 57.8 (CH–COOH), 48.2 (CH2-NH), 39.9 (NH–CH2–
CH2–CH2–CH2–CH2), 36.3 (CH2–C=O), 30.2 (CH2–CH),
41.8 (NH–CH2–CH2–CH2–CH2–CH2), 29.3 (NH–CH2–CH2–
From methotrexate-caproic acid, as in the case of
methotrexate-caproic hydroxamic acid. Yellow crystals
(36%); m.p. 153-155 °C. H NMR (D2O, pD ca. 9) ꢀ: 8.59
1
(1H, s, CH=N), 7.68 d (2H, d, CH=C–C=O), 6.88 (2H, d,
CH=C–NH), 4.55 (2H, s, CH2–NCH3), 4.40 (1H, s, CH–
COOH), 3.18 (3H, s, CH3), 2.85 (2H, t, NH-CH2-CH2–
CH2–CH2-CH2), 2.32 (2H, t, CH2–C=O), 2.11 (2H, t, NH-
CH2-CH2–CH2–CH2-CH2), 1.99 (2H, d, CH2–CH), 1.49 (4H,
m, NH-CH2-CH2–CH2–CH2-CH2, NH-CH2-CH2–CH2–CH2-
CH2), 1.18 (2H, q, NH-CH2-CH2–CH2–CH2-CH2); 13C NMR
(DEPT) (D2O, pD = ca. 10) ꢀ: 151 (CH=N), 131 (CH=C–
C=O), 114 (CH=C–NH), 57.4 (CH2-NCH3), 55.0 (CH–

272 Medicinal Chemistry, 2011, Vol. 7, No. 4
Carrasco et al.
CH2–CH2–CH2), 28.4 (NH–CH2–CH2–CH2–CH2–CH2), 28.1
(NH–CH2–CH2–CH2–CH2–CH2); IR: 1641 cm-1 (ꢀC=O, new
amide bond), 1538 cm-1, 1513 cm-1 (ꢀNH(CONH), new amide
bond); MS (FAB) m/z: 674 (20%) (M+1).
Tert-butyl (4-((6-(hydroxyamino)-6-oxohexyl)carbamoyl)
phenyl)carbamate (Boc-pABA-caproic hydroxamic acid, 12).
From Boc-pABA-caproic acid, as in the case of folate-
caproic hydroxamic acid, but after the evaporation of the
solvent DMF the solid residue was taken into H2O and the
insoluble solid was identified as the product. Recrystalliza-
tion from CH3CN provided the pure product as white crystals
(S)-2-(4-(((2-Amino-4-hydroxypteridin-6-yl)methyl)
amino)benzamido)-5-((4-((6-(hydroxyamino)-6-oxohexyl)
carbamoyl)phenyl)amino)-5-oxopentanoic acid (Folate-
pABA-caproic hydroxamic acid, 4).
1
(88%); m.p. 133 °C. H NMR (MeOD) ꢁ: 7.67 (2H, d,
CH=C-CO), 7.38 (2H, d, NH-C=CH), 3.33 (2H, t, NH-CH2-
CH2–CH2–CH2-CH2), 2.04 (2H, t, NH-CH2-CH2–CH2–CH2-
CH2), 1.58 (4H, m, NH-CH2-CH2–CH2–CH2-CH2, NH-CH2-
CH2–CH2–CH2-CH2), 1.47 (9H, s, C(CH3)3), 1.32 (2H, q,
NH-CH2-CH2–CH2–CH2-CH2). MS (FAB) m/z: 689 (M+1).
From folate-pABA-caproic acid, as in the case of folate-
caproic hydroxamic acid. Yellow crystals (65%); m.p. 260-
270 °C (decomposition). 1H NMR (D2O, pD = ca. 9) ꢁ: 8.57
(1H, s, CH=N), 7.66 (2H, d, NH-C=CH(pABA)), 7.52 d
(2H, d, CH=C–C=O), 7.26 (2H, d, CH=C-CO(pABA)), 6.80
(2H, d, CH=C–NH), 4.58 (2H, s, CH2–NH), 4.40 (1H, s,
CH–COOH), 3.00 (2H, t, NH-CH2-CH2–CH2–CH2-CH2),
2.45 (2H, t, CH2–C=O), 2.11 (4H, m, CH2–CH, NH-CH2-
CH2–CH2–CH2-CH2), 1.35 (6H, br, NH-CH2-CH2–CH2–
CH2-CH2, NH-CH2-CH2–CH2–CH2-CH2, NH-CH2-CH2–
CH2–CH2-CH2); 13C NMR (DEPT) (D2O, pD ca. 9) ꢁ: 150
(CH=N), 132 (CH=C-CO(pABA)), 131 (CH=C–C=O), 124
(NH-C=CH(pABA)), 115 (CH=C–NH), 57.3 (CH–COOH),
48.2 (CH2-NH), 41.8 (NH–CH2–CH2–CH2–CH2–CH2), 39.9
(NH–CH2–CH2–CH2–CH2–CH2), 35.0 (CH2–C=O), 30.6
(CH2–CH), 28.5 (NH–CH2–CH2–CH2–CH2–CH2), 27.9
(NH–CH2–CH2–CH2–CH2–CH2), 27.5 (NH–CH2–CH2–
CH2–CH2–CH2); IR: 1640 cm-1 (ꢀC=O, new amide bond),
1536 cm-1, 1511 cm-1 (ꢀNH(CONH), new amide bond). Anal.
found: C, 55.63; H, 5.3.; N, 20.56%; calculated for
C32H36N10O8: C, 55.81; H, 5.27; N, 20.34%.
4-Amino-N-(6-(hydroxyamino)-6-oxohexyl)benzamide
(pABA-caproic hydroxamic acid, 5).
Boc-pABA-caproic hydroxamic acid (3.01 g, 4.50
mmoles) was dissolved in a mixture of 20 % of trifluoroace-
tic acid (TFA) in CH2Cl2 (40 mL) and was left stirring for 4
h. Then the solvents were evaporated and recrystallization
from methanol provided the pure product as white crystals
1
(22%); m.p. 43 °C. H NMR (D2O) ꢁ: 7.58 (2H, d, CH=C-
CO), 6.83 (2H, d, NH-C=CH), 3.33 (2H, t, NH-CH2-CH2–
CH2–CH2-CH2), 2.05 (2H, t, NH-CH2-CH2–CH2–CH2-CH2),
1.59 (4H, m, NH-CH2-CH2–CH2–CH2-CH2, NH-CH2-CH2–
CH2–CH2-CH2), 1.35 (2H, q, NH-CH2-CH2–CH2–CH2-
CH2); 13C NMR (DEPT) (D2O) ꢁ: 131 (CH=C-CO), 126
(NH2-C=CH), 42.3 (NH–CH2–CH2–CH2–CH2–CH2), 34.0
(NH–CH2–CH2–CH2–CH2–CH2), 30.5 (NH–CH2–CH2–
CH2–CH2–CH2), 27.9 (NH–CH2–CH2–CH2–CH2–CH2), 27.6
(NH–CH2–CH2–CH2–CH2–CH2); IR: 1626 cm-1 (ꢀC=O); MS
(FAB) m/z: 266 (M+1). Anal. found: C, 58.92; H, 7.19; N,
15.97%; calculated for C13H19N3O3: C, 58.85; H, 7.22; N,
16.13%.
4-((Tert-butoxycarbonyl)amino)benzoic acid (Boc-pABA,
10).
A solution mixture of pABA (1.03 g, 7.5 mmol) in water
(8 mL) and 1,4-dioxane (8 mL) was cooled in an ice bath at
0 °C followed by the parallel addition of Na2CO3 (1.53 g, 15
mmol) in water (10 mL) and di-tert-butyl-dicarbonate
(Boc2O) (1.80 g, 8.25 mmol) in 1,4-dioxane (10 mL). The
reaction mixture was stirred for 4 h at 0 °C and then it was
left to heat up to RT overnight. The pH of the solution was
decreased to pH = 2 with 2 M HCl and a white solid was
precipitated as the product, which was filtered off and
washed with large amount of HCl solution to get the pure
HDAC Inhibition Assays
The bioassay for HDAC8 activity was performed using
an HDAC fluorescence activity assay/drug discovery kit
(CycLex® HDAC8 Deacetylase Fluorometric Assay Kit)
according to the manufacturer’s recommendation. The assay
measures the activity of HDAC8 towards a fluoro-substrate
containing acetylated lysine side chains. The buffer for sub-
strate, enzymes and inhibitors was 20 mM Tris·HCl, pH 8.0,
125 nM NaCl, 1% glycerol. Human recombinant HDAC8
(10 μL) enzyme was incubated in buffer for 10 min at 25 °C
in the presence of fluoro-substrate (20 μM), lysyl endopepti-
dase (0.25 mAU/mL) and different inhibitor concentrations
(from 0.1 to 200 μM) in DMSO (5 μL), in a total volume of
50 μL in 96 well flat-bottomed plates. After incubation, the
reactions gave a stable fluorescence signal for up to 60 min.
The fluorescence of each reaction was measured on a
fluorometric reader (Molecular Device SpectraMax Gemini
XS plate reader) at 37 °C with excitation at 340 nm and
emission at 440 nm. The percent inhibition was calculated
from the fluorescence reading of the inhibited reaction rela-
tive to the control reaction with buffer (5 μL) in place of
inhibitor. As reference, control Trichostatin A was used (5
μL, 20 μM) to have a 100% inhibition. All of the assays were
performed in duplicate, and all of the values were used for
calculation of IC50. The HDAC8 inhibition activity was ex-
pressed in relative fluorescent units. Percent of inhibition
1
product as white crystals (95%); m.p. 205-206 °C. H NMR
(acetone-d6) ꢁ: 7.85 (2H, d, CH=C-CO), 7.39 (2H, d, NH-
C=CH), 1.51 (9H, s, C(CH3)3); IR: 3390, 3423, 3484, 3566
cm-1 (ꢀNH).
6-(4-((Tert-butoxycarbonyl)amino)benzamido)hexanoic
acid (Boc-pABA-caproic acid, 11).
From Boc-pABA and 6-amino-caproic acid, as in the
case of folate-caproic acid, but after the evaporation of the
solvent DMF the solid residue was taken into a mixture of
H2O and CH2Cl2 at neutral pH and the insoluble solid was
identified as the product. Recrystallization from methanol
provided the pure product as white crystals (78%); m.p. 143
°C. 1H NMR (MeOD) ꢁ: 7.61 (2H, d, CH=C-CO), 7.35 (2H,
d, NH-C=CH), 3.30 (2H, t, NH-CH2-CH2–CH2–CH2-CH2),
2.14 (2H, t, NH-CH2-CH2–CH2–CH2-CH2), 1.54 (4H, m,
NH-CH2-CH2–CH2–CH2-CH2,
NH-CH2-CH2–CH2–CH2-
CH2), 1.44 (9H, s, C(CH3)3), 1.32 (2H, q, NH-CH2-CH2–
CH2–CH2-CH2).

Novel Folate-Hydroxamate Based Antimetabolites
Medicinal Chemistry, 2011, Vol. 7, No. 4 273
was calculated from control reactions without the inhibitor.
IC50 was determined using the formula: vi/v0 = 1/(1 +
[I]/IC50), where vi is the initial velocity of substrate cleavage
in the presence of the inhibitor at concentration [I] and v0 is
the initial velocity in the absence of the inhibitor. Results
were analyzed using SoftMax Pro software and GraFit soft-
ware [28,29].
binding site. The extracted ligands underwent a conforma-
tional search of 1000 steps in a water environment (using the
generalized-Born/surface-area model) using Macromodel
software [31]. The algorithm used was based on the Monte
Carlo method with the MMFFs force field and a distance-
dependent dielectric constant of 1.0. The ligands were then
minimized using the conjugated gradient method until a con-
vergence value of 0.05 kcal/Å mol was reached, using the
same force field parameters as those used for the conforma-
tional search. The minimized ligands were docked in their
corresponding proteins by means of GOLD 3.0.1 [32]. The
region of interest used by GOLD was defined in such a man-
ner that it contains the residues which stay within 7 Å from
the ligand in the X-ray structures; the zinc ion in HDAC8
was set as trigonal bipyramid atom. The “allow early termi-
nation” command was deactivated while the possibility for
the ligand to flip ring corners was activated. After setting the
remaining Gold parameter with the default values, the
ligands were submitted to 200 genetic algorithm runs. Six
docking analyses were carried out for HDAC8: in the first
three cases, the three fitness functions implemented in
GOLD, GoldScore, ChemScore and ASP, were used. In the
last three, the formation of hydrogen bonds between the ni-
trogen atom of His142 and His143 and the ligands was also
imposed, and the three fitness functions were used again. In
the case of DHFR, only three docking analysis, using the
three fitness functions presented in GOLD, were considered.
The best docking procedure was then used for further stud-
ies. The docking results were evaluated through the compari-
son of the obtained docked positions of the ligand and the
experimental ones. As a measure of docking reliability, the
RMSD between the positions of heavy atoms of the ligand in
the calculated and the experimental structures was taken into
account.
The assay for HDAC activity in crude nuclear extract
from HeLa was performed using an HDAC fluorescence
activity assay/drug discovery kit (CycLex® HDACs Deacety-
lase fluorometric assay kit, CY-1150), according to the
manufacturer’s recommendation. Crude nuclear extract from
HeLa (10 μL) was incubated in buffer for 10 min at 25 °C in
the presence of fluoro-substrate (20 μM), Lysyl endopepti-
dase (0.25 mAU/mL) and varying concentrations of inhibitor
(from 0.1 to 100 μM) in DMSO (10 μL), in a total volume of
50 μL in 96 well flat-bottomed plates. The fluorescence of
each reaction was measured at 37 °C with excitation at 355
nm and emission at 460 nm and data were analyzed as re-
ported above.
Assays of DHFR Activity
L. casei DHFR activity, in the presence and in the ab-
sence of inhibitors, has been assayed spectrophotometrically
by monitoring the decrease in absorbance at 340 nm due to
oxidation of NADPH. All assays were performed at 30 °C in
a Shimadzu 2401PC double-beam spectrophotometer. The
reaction mixture contained 100 μM of dihydro-folic acid,
150 μM of NADPH and 2.5 μg/mL purified DHFR. The re-
action was started by the addition of the enzyme (1-2.5 ꢀg)
in a final volume of 1.0 mL and read against a blank cuvette
containing all components except the enzyme. Synthesized
compounds were added in the reaction mixture to final con-
centrations 0.1-1000 ꢀM. For a reference inhibition curve,
MTX was added into reaction mixture to final concentrations
of 0.001-0.1 ꢀM.
The molecular structures of 1-5 were built, parameterized
and energy minimized with Maestro [30]. These ligands were
docked into HDAC8 and DHFR active sites using GOLD
software and the procedure described above was followed.
To perform the docking calculations, the same conditions
and parameters which displayed the best prediction of the
binding mode of the inhibitors were employed, namely
GoldScore scoring function for both enzymes.
Cell Culture Studies
The lung carcinoma cell line A549 was obtained from
American Type Culture Collection. The prostate carcinoma
cell line PC3 was a kind gift from Dr. James Norris, Medical
University of South Carolina. Cells were maintained in
RPMI-1640 supplemented with 10% heat-inactivated fetal
bovine serum, 2 mM glutamine and 1 mM sodium pyruvate
(complete medium). All cells were grown at 37 ºC under
humidified air containing 5% CO2. Cells were plated in 96-
well plates at a density of about 5000 cell/well. Treatment
with different concentrations of each inhibitor was per-
formed constantly for 72 h. MTT cell proliferation assay was
performed using CellTiter 96 kit (Promega), according to
manufacturer's directions.
ACKNOWLEDGEMENTS
The authors would like to thank the Portuguese Funda-
ção para a Ciência e Tecnologia for finantial support. We
also thank the Portuguese NMR and MS networks (IST-UTL
Center), created by the Portuguese Foundation for Science
and Technology (FCT), for providing access to their facili-
ties.
Docking Studies
REFERENCES
The HDAC8 (PDB code 1T69) and DHFR (PDB code
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from the active site, hydrogen atoms were added by means
of Maestro 7.5 [30,31] using the all-atom model. To assess
the reliability and validity of the docking procedure used, the
bound ligands were flexibly re-docked into the respective
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Received: November 13, 2010
Revised: February 01, 2011
Accepted: March 06, 2011