Design and synthesis of novel 3,4-disubstituted pyrazoles for nanomedicine applications against malignant gliomas

Article abstract of DOI:10.1016/j.ejmech.2010.01.014

A series of novel 3,4-disubstituted pyrazoles were synthesized. The cytotoxicity against U87MG glioma cell line have been investigated in vitro and three of these compounds showed promising inhibitory activity on cell growth with an IC₅₀ lower

Full text of DOI:10.1016/j.ejmech.2010.01.014

European Journal of Medicinal Chemistry 45 (2010) 2024–2033
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design and synthesis of novel 3,4-disubstituted pyrazoles for nanomedicine
applications against malignant gliomas
,
a
a
a
Mauro Comes Franchini a , Bianca Flavia Bonini , Carlo Maurizio Camaggi , Denis Gentili ,
*
Annalisa Pession b, Monica Rani b, Elena Strocchi a
a
`
Dipartimento di Chimica Organica, Universita di Bologna, Viale Risorgimento 4, Bologna 40136, Italy
b Dipartimento Clinico Scienze Radiologiche ed Istocitopatologiche, Ospedale Bellaria, Via Altura 3, Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 3,4-disubstituted pyrazoles were synthesized. The cytotoxicity against U87MG glioma
Received 23 July 2009
Received in revised form
8 January 2010
Accepted 11 January 2010
Available online 20 January 2010
cell line have been investigated in vitro and three of these compounds showed promising inhibitory
activity on cell growth with an IC50 lower than 90
receptor (TGF- -RI; PDB: 1py5) has been done for lead optimization of the mentioned compounds as
potential TGF- -RI1 inhibitors. In particular, 3-aryl-4-amido pyrazole containing long -amino-aliphatic
mM. AutoDock molecular docking into type I TGF-
b
b
b
u
chain emerged as a good candidate for further optimization. Entrapment into targetable PEG-based
micelles improved growth inhibition IC50 values up to 100 nM and this could lead to a novel drug
delivery strategy for treating glioblastoma.
Dedicated to the memory of my nephew
Veronica (M.C.F.)
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Pyrazoles
Glioblastoma
Drug delivery
Nanomedicine
Docking
1. Introduction
and suppression of the immune response. Malignant gliomas retain
expression of essential elements of the TGF-b signal transduction
The incidence of primitive brain tumors increased in the last
decades and tumors of the astrocytic series are the most frequent.
Glioblastoma (GBM), the astrocytic tumor of the highest grade,
remains almost universally fatal despite new diagnostic tools and
maximal therapy [1]. Gliomas are often characterized by resistance
to the conventional chemo- or radio-therapy and therefore, it is
important to search innovative and powerful therapeutic tools such
as the molecular targeting, to inhibit specific pathways that play
a role in glioblastoma.
pathways. Therefore, specific inhibition of this pathway could offer
a novel therapy for the treatment of malignant gliomas. Recent
studies have shown that several small heterocycles are inhibitors of
the type I TGF-b receptor (TGF-b-RI) [4]. In all these cases, the
small-molecule ATP-competitive contained a central heterocyclic
scaffold and a key functionality, ‘‘warhead’’ group, necessary for
potent binding to the kinase domain active site. Imidazoles such as
SB-431542 [2,5], have been tested on a panel of human malignant
glioma cell lines and effectively inhibited human cells in vitro and in
vivo. Also pyrazoles have proved to be inhibitors of the TGF-
Transforming growth factor-
signal transduction pathways, plays a critical role in malignant
gliomas [2]. TGF- is a multifunctional cytokine that promotes cell
growth and malignant gliomas express TGF- ligands and recep-
tors, suggesting the presence of autocrine or paracrine loops [3].
Furthermore, in normal epithelial tissues, TGF- inhibits cell
b (TGF-b) pathway, among the
b
receptors kinase domain, showing good in vitro activity [6].
b
The past years have witnessed a great development of nano-
b
medicine platforms, which included drug delivery systems in the
nanometer size range (preferably 1–100 nm) containing encapsu-
lated, dispersed, adsorbed or conjugated drugs and/or imaging
agents [7]. Perhaps the greatest impact [8] of nanotechnologies in
cancer therapy, up to now, is the new concept of drug delivery, since
several obstacles may be overcome. For example, many drugs that
are not very soluble, can be solubilized by formulating them into
various type of nanosuspensions and kept in circulation for longer
periods. Drug toxicity is reduced as a consequence of preferential
b
growth whereas, in advanced epithelial cancer, it promotes tumor
growth through induction of tumor invasion and neoangiogenesis
* Corresponding author. Tel.: þ39 0512093626; fax: þ39 0512093654.
E-mail address: mauro.comesfranchini@unibo.it (M. Comes Franchini).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.014

M. Comes Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 2024–2033
2025
accumulation at target sites and lower concentration in healthy
tissues. In the case of brain cancers, many drugs have difficulty in
crossing the blood–brain barrier (BBB) to attack the tumor; drug-
loaded nanoparticles are able to penetrate this barrier, and have
been shown to greatly increase therapeutic concentrations. More-
over, it has been also demonstrated that the BBB may be overcome
by particles with a size dimension of about 50 nm or by lipid-
mediated transport or receptor mediated and polyethylene glycol
(PEG) assisted processes [9]. Most nanosized delivery systems are
made up of micellar amphiphilic polymers that consist of PEG and
a low molecular-weight hydrophobic core-forming block such as
poly(lactic)-co-glycolic acid (PLGA) or poly(lactic)acid (PLA). The
hydrophobic core serves as a natural carrier environment for
hydrophobic drugs and the hydrophilic shell allows particle stabi-
lization in aqueous solution [10].
(DMAP) in CH2Cl2 for 48 h giving amide 5, after Boc removal, with
64% overall yield. Long-chain aliphatic amides having -functional
u
groups were also prepared: reaction of 1 with 11-mercaptounde-
canoic acid and 12-(tert-butoxycarbonylamino)dodecanoic acid in
CH2Cl2 with DCC and DMAP gave 6 in 53% yield and, after Boc
removal, 7 in 75% overall yields. Reaction with 12-hydroxy-
dodecanoic acid under the same reaction conditions gave 8 in 41%
yield.
Replacement of the phenyl in C-3 with a pyrid-2-yl group was
then carried out in order to make a comparison in the inhibitory
activity. In this case the synthetic sequence started with the
cyclocondensation of 3-dimethylamino-1-pyridin-2-yl-propenone
[12] with methyl hydrazine adapting a patent publication [13] to
form a 1:1 mixture of the two possible regioisomers 1-methyl-3-
(pyrid-2-yl)-1H-pyrazole and 1-methyl-5-(pyrid-2-yl)-1H-pyrazole
which were separated by column chromatography on silica. The
desired compound with the 2-pyridyl group in C-3, after identifi-
cation by nuclear overhouser effect (NOE) experiments, was
nitrated and reduced to give 4-amino-1-methyl-3-(pyridin-2-yl)-
1H-pyrazole 9 with 31% overall yield.
We report here the in vitro cellular-based assay screening of
a small library of novel 3,4-disubstituted pyrazoles and the study of
their inhibitor effect on growth of U87MG, a stabilized cell line
derived from human glioma expressing TGF-b-RI. We also present
our nanomedicine applications to formulate some selected
3,4-disubstituted pyrazoles in a delivery system such as nanosized
PLGA-b-PEG polyester micelles. We thought that 3,4-disubstituted
pyrazoles could have been chosen as a platform for SAR develop-
ment according to Sawyer’s suggestion [6a], who reported that the
minimum requirements for tight binding at the active site of the
ATP consist of the presence of a 2-pyridyl group on the 3-position of
the pyrazole-based ring and a substituent at the 4-position
featuring a hydrogen-bond acceptor [6a]. Our pyrazoles were
designed with a simple aryl group on the C-3 and with an amide
moiety RCONH– on the C4 of the heterocyclic ring. A molecular
docking study was performed to characterize the binding affinity of
Also in this case, the building block 9 was amidated with acyl
chlorides or with carboxylic acids as in the case of 1 to give
compounds 10–16 with satisfactory overall yields (Scheme 2).
3. Results and discussion
We tested with the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) test (see Experimental), the
in vitro inhibitory activity of the pyrazoles 2–8 and 10–16 (diluted in
DMSO) on the growth of U87MG glioblastoma human cell line. In
Table 1 the IC50 values, evaluated after 24 h of incubation, have
been reported, with most of the pyrazoles showing an inhibitory
putative lead candidates intoTGF-b-RI ATP binding site. The growth
inhibitory activities of the synthesized pyrazole derivatives against
glioma cells were investigated.
activity at concentration higher than 150
active molecules, we have found compound 3, 4, 6, 7 and 15 which
gave IC50 in the range 77–90 M (see entries 2, 3, 5, 6 and 13). No
mM. Among the more
m
significant changes have been observed after 48 h.
2. Chemistry
3.1. Molecular docking study
The building block 4-amino-1,3-diphenyl pyrazole
prepared with slight modification of literature [11].Reaction
of -bromoacetophenone with phthalimide potassium salt in
N,N-dimethylformamide (DMF) gave the -phthaloylamino aceto-
1 was
Binding modes and binding affinities of the synthesized
compounds within the ATP pocket of the TGF-b-RI kinase were
a
a
examined using the AutoGrid 4.0 and AutoDock 4.01 programs. The
ADT 1.5.1 package was used for visualization of the results (see
experimental section for the procedure for molecular docking) [14].
phenone in 89% yield. Subsequent one-pot reaction with neat
dimethylformamide dimethyl acetal (DMFDMA) followed by
phenyl hydrazine at reflux for 3 h gave the 4-N-Pht protected
1,3-diphenyl pyrazole with 59% overall yield. Deprotection with
hydrazine in refluxing ethanol gave 4-amine-1,3-diphenyl-
1H-pyrazole 1 in 80% yield (Scheme 1).
3.2. Experimental section for the molecular docking procedure:
analysis of the binding mode
The reaction of 1 with the benzoyl-, o-methoxybenzoyl- and
dodecanoyl-chloride gave the desired aromatic and aliphatic
amides 2–4 in the range 65–81% yields after column chromato-
graphy followed by crystallization. We have also prepared an
The conformation with the lowest (docked) energy was
considered to be the ‘‘best’’ docking result. We analysed the loca-
tion and orientation of the synthesized compounds and the inter-
actions into the binding site. In each case, the substituted
heterocyclic scaffold entered into the binding pocket site with good
binding affinity. The hydrophobic aryl moieties fit tightly into the
enantiopure
Thus commercially available
dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine
L-amino acid-derived amide of the building block 1.
L-Boc-alanine was reacted with 1 and
O
2: R=C6H5
3: R=o-MeO-C6H4
4: R=C11H23
O
O
Ph
Ph
N
R
NH
PhtN
NPht
b,c
Ph
Br
H2N
d
e
a
5: R=CH3(NH2)CH
6: R=HSC10H20
7: R=NH2C11H22
8: R=HOC11H22
N
N
1
N
N
N
Ph
Ph
Ph
Scheme 1. Reagent and conditions: (a) NKP, DMF rfx, 24 h, 89%. (b) DMFDMA, 100 ꢀC, 18 h, 78%. (c) PhNHNH2, rfx, 3 h, 76%. (d) NH2NH2 EtOH, rfx, 1 h, 80%. (e) RCOCl, EtN(iPr)2,
CH2Cl2, rt, 24 h or RCOOH, DCC–DMAP, CH2Cl2, rt, 48 h. For Boc removal: HCl/AcOEt, rt, 3 h.

2026
M. Comes Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 2024–2033
O
10: R=C6H5
NMe2
b
O
O
N
11: R=o-MeO-C6H4
12: R=C11H23
N
N
R
NH
H2N
c,d
a
e
N
13: R=CH3(NH2)CH
14: R=HSC10H20
15: R=NH2C11H22
16: R=HOC11H22
N
N
N
N
N
N
N
CH3
9
CH3
CH3
Scheme 2. Reagent and conditions: (a) DMFDMA, EtOH, 6 h, rfx, 58%. (b) MeNHNH2, 2-methoxyethanol, 3 h, rfx, 64% overall for two isomers. (c) HNO3–H2SO4, 48 h rt to 100 ꢀC 1 h;
H2, Pd–C, EtOH–DMF, 24 h, rt overall 31%. (e) RCOCl, EtN(iPr)2, CH2Cl2, 24 h, rt or RCOOH, DCC–DMAP, CH2Cl2, rt, 48 h. For Boc removal: HCl/AcOEt, 3 h, rt.
hydrophobic pockets of the ATP binding site. All the examined
compounds revealed a common binding mode in the adenosine
cleft through a complementary mainly apolar interaction surface.
Residues lining the binding pocket (within 3.5 Å of the ligand) were
LYS213, GLY214, VAL219, ALA230, VAL231, LYS232, LEU278, SER280,
LEU340 and ASP351. No relevant hydrogen-bonding interaction
was observed, with the exception of compounds 6 and 7, showing
the interaction between the terminal amino or SH group with
ASP290 (see Fig. 1). Therefore, in our docking results the aromatic
parts of the molecules seemed to be more determinant for a good
interaction between ligands and macromolecule. Compounds 6, 7
and 3 (Table 1, Fig. 1) resulted as best docked molecules inside the
ATP site pocket of the target receptor with a good binding affinity
(binding free energy [BE, DG docking energy] of ꢁ10.25 kcal/mol,
ꢁ10.22 kcal/mol and ꢁ10.12 kcal/mol respectively and calculated
inhibition constant [KI, derived from AutoDock binding energy] of
30.85, 32.47 and 38.50 nM respectively).
Pyrazoles with phenyl substituents at N-1 and C-3 of the
pyrazole-based moiety and lateral alkyl chain or phenyl substituent
in the amidic R–CO–NH exhibited better values of binding free
energies with DG from ꢁ10.22 to ꢁ9.35 kcal/mol and KI from
32.47 nM to 139.70 nM (Table 1).
A long alkyl lateral chain facilitated the molecule orientation
into the binding site and a terminal NH2 or SH group on the chain
allow the molecule to form an H bond with ASP290 residue at the
entrance of the binding site. The compounds with a methyl group at
N1 position and a pyrid-2-yl on C-3, such as molecules 10–16
showed lower potential affinity than the others, with the exception
of 15 (probably due to the ending amino functionality). Unfortu-
nately, compound 6 showed insolubility and instability in several
organic solvents, this resulted in a crucial aspect for the formulation
into micelles, therefore we did not proceed further with 6. The
observed experimental IC50 are about 100 times higher than
calculated constants of inhibition. This could be explained by the
absorption process which the compounds should undergo before
the Kinase inhibition suggesting that more lipophilic derivatives,
with a favourable partition coefficient at biological pH, could enter
more easily and show better activity since the ATP binding site of
TGF-b-RI is located inside the cells.
Thioamides are important because of their structural similarity
to the amide bond and their potential gain of in vivo stability [15].
Of particular importance could be the hydrogen-bonding ability of
the thioamide group [16]; its NH is more acidic, and thus a stronger
hydrogen-bond donor, while the S-atom is generally considered
a weaker acceptor, although there is also evidence that the
H-bonding-acceptor capacity of the thioamide S-atom may equal
that of the amide O-atom [16]. Accordingly and based on the above
considerations, we decided to transform 7 into the corresponding
thioamide. Thionation of 17 was accomplished with Lawesson’s
reagent under standard conditions in toluene at 55 ꢀC (Scheme 3),
giving 18 which was Boc-deprotected under acidic conditions to
give, in 89% overall yield, the 12-amino-N-(1,3-diphenyl-1H-pyr-
azol-4-yl)dodecanethioamide 19.
Virtual docking of compound 19, did not show however any
hydrogen-bonding interaction between thioamide group and
binding site of macromolecule and calculated BE was worst than
the corresponding value for amide compound 7 (BE ꢁ9.72 kcal/mol,
KI 75.6 nM); finally in vitro IC50 for 19 (diluted in DMSO) was found
to be 82 mM which is in line with other compounds 3, 4, 6 and 7.
We selected compounds 3, 7 and 19 for the formulation into
polyester micelles. We then moved to the synthesis of the block co-
polymer to provide the biodegradable matrix for the encapsulation
and sustained release of 3, 7 and 19. Following a previous report
[17], PLGA-b-PEG was synthesized by using a two-step reaction in
anhydrous organic solvents.
Table 1
BE, KI, and IC50 values for pyrazoles 2–8 and 10–16.
Entry
Product
BE
Calculated KI
M)
Experimental
IC50 (mM)
a
(kcal/mol)
(
m
1
2
3
4
5
6
7
8
2
3
4
5
6
ꢁ9.84
ꢁ10.12
ꢁ9.55
ꢁ9.79
ꢁ10.25
ꢁ10.22
ꢁ9.35
ꢁ7.77
ꢁ8.09
ꢁ8.56
ꢁ8.24
ꢁ8.18
ꢁ8.21
ꢁ8.40
0.061
0.039
0.099
0.067
0.031
0.032
0.140
2.02
>150
81
81
>150
90
77
>150
>150
130
>150
>150
>150
90
7
8
10
11
12
13
14
15
16
9
1.18
10
11
12
13
14
0.534
0.917
1.02
0.961
0.700
Fig. 1. Best docking conformation of the molecules 3 (blue), 6 (yellow) and 7 (pink)
into the ATP binding site of TGF-b-RI (the binding pocket is shown in solid surface).
>150
BE: binding free energy; KI: calculated inhibition constant.
IC50 values are means of three different experiments; assay details are described
in the Experimental section.
H-bond of terminal amino-group with ASP290 residue of the macromolecule is also
shown. (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of this article.)
a

M. Comes Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 2024–2033
2027
O
S
S
NH
Ph
NH
NH
Ph
Ph
b
a
( )11
BocHN
( )11
( )11
H2N
BocHN
N
N
N
N
Ph
N
Ph
N
17
18
19
Ph
Scheme 3. Reagent and conditions: (a) Lawesson’s reagent, Toluene, 55 ꢀC, 92%. (b) HCl/AcOEt, 3 h, rt, 97%.
The carboxyl-capped PLGA–COOH (inherent viscosity 0.12 dl/g,
MW w7 kDa) was activated with N-hydroxysuccinimide (NHS)
using dicyclohexylcarbodiimide (DCC) coupling chemistry and then
conjugated to the amine monofunctional PEG–NH2 (MW w2 kDa),
in chloroform and in the presence of diisopropylethylamine
(DIPEA), this coupling efficiency being determined by 1H NMR
(Scheme 4).
Pyrazole-encapsulated PLGA-b-PEG nanoparticles (NPs) were
prepared by using nanoprecipitation method [18]. Taking advan-
tage of a previous study about the solubility [18b], we selected the
DMF as solvent using a solvent:water ratio of w0.1 and a polymer
concentration of 50 mg/mL, the pyrazole loading was 2% by weight
relative to the polymer (Scheme 5).
possibly because of a more efficient uptake of polymeric containing
drug nanoparticles blocking P-gp systems, led to a significantly
enhanced drug concentration within tumor cells.
Nanoparticle PLGA–PEG–7 showed the best inhibitory trend as
confirmed by the data reported in Fig. 3(A). The curves show the
percentage decrease of cell viability after 48 h incubation with the
three NPs containing products at different concentrations. Fig. 3(B)
reports the results of more active compound after 24 and 48 h of
drug incubation. The cell images reported down in Fig. 3 show
control U87MG cells without compound incubation (A) and after
48 h incubation with IC50 concentration (B), confirming the
cytoxicity of the compound.
The determination of particles sizes and polydispersities were
measured by dynamic light scattering (DLS) as shown in Fig. 2 and
proved to be 86.72 for PLGA–PEG–3, 87.15 for PLGA–PEG–7 and
87.51 for PLGA–PEG–19 (in Fig. 2 only data for PLGA–PEG–7 are
shown). PDI measurements, being in the range 0.12–0.13, reveal
a narrow size distribution for all three cases. The 3, 7 and 19 percent
contents into the polymeric matrix were measured with UV-anal-
ysis based on absorbance intensity compared to standard absorp-
tion curve of the pyrazoles and were found to be 15%, 27% and 28%
respectively for 3, 7 and 19 (see Fig. 2).
Inhibitory activity on the cell growth (U87MG human cell line)
was tested after 24 and 48 h of incubation, taking into account
additional time necessary for drug release from micelles (Table 2).
The inhibitory effect of PLGA–PEG–7 and PLGA–PEG–3, was already
better after 24 h of incubation than the corresponding effect
observed with free 3 and 7 compounds (see Table 1) while
4. Conclusion
A series of 3,4-disubstituted pyrazoles were synthesized and
evaluated as potential inhibitors against U87MG glioma cell lines
expressing TGF-b-RI. Based on the interesting values reported by
Sawyer with pyrazoles having a pyrid-2-yl on C-3, our simpler
3-phenyl derivatives, showed preliminary satisfactory activity.
Moreover, the concept of an amide with long
aliphatic chain on the C-4 position seems also to be promising.
Considering TGF- -RI as a potential target, docking evaluation
indicated as most potentially effective inhibitory compound to the
ATP TGF- -RI binding site those with the following structural
u-functionalised
b
b
features: (1) hydrophobic aryl moiety; (2) pyrazole-based moiety
with phenyl substituents at N1 and C-3 and lateral alkyl chain or
phenyl substituents in the R–CO–NH. The common binding mode of
all derivatives into the ATP binding site through a complementary
apolar interaction surface, and no relevant hydrogen-bonding
interaction, suggested that the aromatic parts of the molecules
seem to be crucial for a good affinity of ligands and macromolecule.
Evaluation of in vitro enzymatic activity, as well as studies on the
inhibition of downstreaming components is also under investiga-
tion. In vitro study confirmed inhibitory effect against the U87MG
glioma cell line at micromolar concentration, and a promising value
of 100 nM has been obtained with PLGA–PEG–7. Moreover, the
compound entrapment into water soluble polyester micelles gave
improved IC50 values, and it seems to be suitable [21] for future in
vivo nanosized drug delivery applications and calls for other studies
which will be reported in due course.
PLGA–PEG–19 gave a 120 mM IC50 value. In Fig. 3(A) the inhibitory
activity at 48 h of the water soluble micelles containing the three
molecules is reported, because of the lower observed IC50 values.
The PLGA–PEG–7 showed the higher activity at all the concentra-
tion tested, with an interesting 100 nM IC50; PLGA–PEG–3 IC50 does
not change after longer incubation, while PLGA–PEG–19 become
more active along the time, giving a 10 mM IC50 value after 48 h.
It has been observed that polymeric nanoparticles containing
chemical drug resulted in an increased cytotoxic effect compared to
free drug [19]. PLGA–PEG nanocapsules developed by us exhibit
a very small size facilitating an intracellular uptake and P-gp
inhibiting properties, since nanoparticles constituents has been
documented can inhibit transmembrane P-glycoprotein (P-gp)
transporting systems. They contribute to the MDR phenomenon in
cancer cells [20], by eliminating the penetrating drug from inside
the targeted tumor cells. Our results showed that in all cases
polymeric nanoparticles containing chemical drugs (product
PLGA–PEG–3, –19 and –7) resulted in an increased cytotoxic effect
compared to that observed for the corresponding free drugs [19],
5. Experimental
5.1. General information
Melting points were determined with a Bu¨chi melting point
apparatus. 1H NMR and 13C NMR spectra were recorded using CDCl3
or CD3OD or DMSO solutions at 300, 400 and 600 MHz for 1H and
75.46, 100.6 and 150.92 MHz for 13C. Chemical shifts (
d) are
O
O
H
N
a, b
OH
reported in ppm relative to CHCl3
(
d
¼ 7.26 for 1H and
d
¼ 77.0 for
O
OMe
O
O
O
o
O
n
13C). J values are given in Hz. 1H NMR and 13C NMR spectral
assignments were made by DEPT, gCOSY and gHSQC experiments.
IR spectra were recorded on a Perkin–Elmer model 257 grating
spectrometer. Mass spectra (MS) were obtained with an electro-
spray ionization source (ESIMS). All the ESIMS spectra were
m
m
n
CH3
O
CH3
O
Scheme 4. Synthesis of the block co-polymer PLGA-b-PEG. Reagent and conditions: (a)
NHS/DCC, DCM, rt, 20 h. (b) DIPEA, PEG–NH2, CHCl3, rt, 18 h. o.y. from PLGA–
COOH ¼ 80%.

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M. Comes Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 2024–2033
Scheme 5. Encapsulation of selected pyrazoles 3, 7 and 19 into PLGA-b-PEG nanoparticles (NPs).
performed using MeOH as the solvent. High Resolution Mass
Spectra (HRMS) were recorded on a micromass LCT spectrometer
using electrospray (ESþ) ionization techniques. Reactions were
conducted in oven-dried (120 ꢀC) glassware under a positive Ar
atmosphere. Transfer of anhydrous solvents or mixtures was
accomplished with oven-dried syringes/septum techniques. THF
was distilled from sodium/benzophenone just prior to use and
stored under Ar. Toluene was distilled from sodium. Et2O was
distilled from phosphorus pentoxide. CH2Cl2 was passed through
basic alumina and distilled from CaH2 prior to use. Other solvents
were purified by standard procedures. Light petroleum ether refers
to the fraction with bp 40–60 ꢀC. The reactions were monitored by
TLC performed on silica gel plates (Baker-flex IB2-F). Column
chromatography was performed with Merck silica gel 60 (70–230
mesh). Preparative thick layer chromatography was carried out on
glass plates using a 1 mm layer of Merck silica gel 60 Pf 254. All
chemicals were used as obtained or purified by distillation as
needed. N-hydroxysuccinimide, N,N0-dicyclohexylcarbodiimide
and N,N-diisopropylethylamine were purchased from Sigma–
search over 1,000,000 conformations, Alchemy2000, Tripos inc,
St. Louis, MO (USA)) followed by semi-empirical AM1 geometry
optimization of the minimum-energy conformation (ArgusLab 4.0,
Thompson MA, Planaria Software LLC, Seattle, http://www.
ArgusLab.com). Preparation of target macromolecule: the target
protein TGF-b receptor (PDB: 1py5) was retrieved from the Protein
Data Bank (http:/www.rcsb.org/pdb/). Polar hydrogen atoms were
added to both macromolecule and ligands, non-polar hydrogen
atoms merged and Gasteiger charges computed and using the ADT
standard procedures. All possible torsions in the ligand (except for
amide bonds) were detected and allowed to freely rotate during
docking.
5.2.2. Procedure for molecular docking
Docking calculations were carried out using AutoDock 4.01 and
AutoGrid 4.0 [14] on a dual-xeon T7400 Dell workstation. Grids
(one grid for each atom type in the ligand, plus an electrostatic and
a desolvation map) were centred on the binding site and were
chosen to be large enough (70 ꢂ 60 ꢂ 60 Å) to allow the ligand to
rotate freely, even in its most fully-extended conformation.
Docking was performed using the AutoDock empirical free
energy function and the Lamarckian genetic algorithm with local
search. Lamarckian genetic algorithms can handle ligands with
more degrees of freedom than the simulated annealing method.
One hundred docking runs with 2,500,000 energy evaluations for
each run were performed for each molecule. Cluster analysis
(RMS tolerance equal to 0.5 Å) was then carried out on the docked
results. Inhibitors were compared according to the lowest docked
energy found. The inhibition constants – KI – were calculated from
the docked energy (Table 1).
Aldrich (St. Louis, MO). Poly(D,L-lactide-co-glycolide) (50/50) with
carboxylic acid end group (PLGA–COOH, inherent viscosity 0.12 dl/
g, MW w7 kDa) was purchased from Lakeshore Biomaterials
(Birmingham, AL, USA). Polyethylene glycol with amino end group
(MeO–PEG–NH2, MW w2 kDa) was purchased from Rapp Polymere
GmbH (Tu¨bingen, Germany).
All reagents were analytical grade or above and used as received.
5.2. Molecular docking
5.2.1. Preparation of molecule
Before starting the docking calculations ligand geometries were
optimised first through a conformational search (Monte-Carlo
5.3. Chemistry
Compound 1,3-diphenyl-1H-pyrazol-4-amine
1
has been
synthesized according to Ref. [11a]. The only difference from the
literature procedure was the purification, in the last step, of the
amine. In our procedure, chromatography on silica with petroleum
ether/EtOAc 1/1 as eluent gave the amine 1 in pure form. The
synthesis of product 9 was performed as follows:
5.3.1. 1-Methyl-3-(pyrid-2-yl)-1H-pyrazole
3-(Dimethylamino)-1-(pyrid-2-yl)prop-2-en-1-one
300 mg
(1.7 mmol) was dissolved in 2-methoxyethanol (5 mL) and heated
to reflux under argon with 83 mg (1.8 mmol) of methyl hydrazine
Table 2
Inhibitory effect expressed as IC50 values for the three nanoparticles PLGA-b-PEG-3,
-7 and -19.
Nanoparticles
IC50 – 24 h
IC50 – 48 h
Free drug
IC50 – 24 h
PLGA–PEG–3
PLGA–PEG–7
PLGA–PEG–19
23
38
120 mM
m
m
M
M
30
0.1
10
m
m
M
M
81
77
82
m
m
m
M
M
M
Fig. 2. Size distribution from Dynamic Light Scattering (DLS) measurement of PLGA-b-
PEG-7 and straight calibration at 255 nm for the determination of 7 into the block co-
polymer.
mM

M. Comes Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 2024–2033
2029
Fig. 3. Inhibitory activity of the three nanoparticles on U87MG human glioma cell line: (A) effect of 3-, 7- and 19-encapsulated PLGA-b-PEG on U87MG cell line after 48 h of
incubation. (B) Effect of the most effective 7-encapsulated PLGA-b-PEG NPs at different times.
for 6 h. After cooling to room temperature, the solvent was
removed in vacuum. The 1H NMR of the crude revealed the pres-
ence of two regioisomers in a ratio 1:1. The crude was chromato-
graphed on SiO2 (petroleum ether/EtOAc 1/1 as eluent). The first
fraction (Rf 0.33) was identified from NOE experiments as 1-
methyl-5-(pyrid-2-yl)-1H-pyrazole. 1H NMR (300 MHz, CDCl3):
4.32 (s, 3H, NMe), 6.67 (d, J ¼ 2.1 Hz, 1H, CH]CH–NMe), 7.33 (dd,
J ¼ 8.0, 4.9 Hz, 1H, Ar–H), 7.60 (d, J ¼ 2.1 Hz,1H, CH]CH–NMe), 7.66
(t, J ¼ 8.0 Hz, 1H, Ar–H), 7.83 (d, J ¼ 7.9 Hz, 1H, Ar–H), 8.76 (d,
J ¼ 4.9 Hz, 1H, Ar–H). 13C NMR (100 MHz, CDCl3): 39.0, 106.1, 121.9,
122.3, 136.3, 137.6, 140.8, 148.7, 149.5. MS m/z: 175 [M þ H]þ, 197
[M þ Na]þ. The second fraction (Rf 0.16) resulted from NOE exper-
iments the 1-methyl-3-(pyrid-2-yl)-1H-pyrazole. Total yield
174 mg (64%). 1H NMR (300 MHz, CDCl3): 3.95 (s, 3H, NMe), 6.84 (d,
J ¼ 2.3 Hz, 1H, CH¼CH–NMe), 7.16 (dd, J ¼ 7.8 Hz, J ¼ 5.0 Hz, 1H, Ar–
H), 7.38 (d, J ¼ 2.3 Hz, 1H, CH]CH–NMe), 7.68 (t, J ¼ 7.9 Hz, 1H, Ar–
H), 7.88 (d, J ¼ 8.0 Hz, 1H, Ar–H), 8.60 (d, J ¼ 4.9 Hz, 1H, Ar–H). 13C
NMR (100 MHz, CDCl3): 38.8, 104.0, 119.5, 121.9, 131.2, 136.2, 149.0,
151.3, 151.9. MS m/z: 160 [M þ H]þ, 183 [M þ Na]þ.
5.2 Hz, 1H, Ar–H), 7.64 (t, J ¼ 8.0 Hz, 1H, Ar–H), 7.93 (d, J ¼ 8.0 Hz,
1H, Ar–H), 8.51 (d, J ¼ 5.2 Hz, 1H, Ar–H). 13C NMR (100 MHz, CDCl3):
39.2, 118.4, 118.9, 120.6, 131.0, 136.1, 142.6, 148.1, 154.6. MS m/z: 175
[M þ H]þ, 197 [M þ Na]þ.
5.3.4. General procedure for the synthesis of compounds 2, 3, 4, 10,
11, 12
The appropriate acylchloride (0.6 mmol) in DCM (3 mL) was
slowly added at 4 ꢀC (ice-water) to a stirred mixture of N,N-diiso-
propylethylamine (1.2 mmol) and product 1 or 9 (0.5 mmol) in
DCM (4 mL). After one night at room temperature, the solvent was
evaporated water was added and the mixture was extracted with
EtOAc. The organic layer was washed with 1 N NaOH and then with
water then dried. Evaporation of the solvent gave a residue that was
chromatographed on SiO2 (petroleum ether/EtOAc 1/1).
5.3.4.1. N-(1,3-Diphenyl-1H-pyrazol-4-yl)benzamide 2. Yield 80%
after crystallization from EtOH. Mp: 212–214 ꢀC. IR (CCl4,
n
¼ cmꢁ1):
3368, 3063, 1669, 1592, 1570, 1542, 1514, 1477, 1446, 1389, 1296,
1259, 1233. 1H NMR (300 MHz, CDCl3): 6.94 (d, J ¼ 8.6 Hz, 1H, Ar–
H), 7.15 (t, J ¼ 7.3 Hz, 1H, Ar–H), 7.20–7.35 (m, 9H, Ar–H), 7.40–7.55
(m, 2H, Ar–H), 8.32 (t, J ¼ 8.1 Hz, 2H, Ar–H), 8.70 (s, 1H, ]CH–N),
9.90 (s, 1H, NH). 13C NMR (100 MHz, CDCl3): 113.25, 121.8–122.8
(3C), 124.9, 126.3, 129.7–129.7 (4C), 132.0, 133.7, 134.6, 139.7, 158.5,
163.7. (MS ESIꢁ): 338 [M ꢁ H]ꢁ. Anal. Calcd for C22H17N3O: C, 77.86;
H, 5.05; N, 12.38. Found: C, 77.80; H, 5.02; N, 12.35.
5.3.2. 1-Methyl-4-nitro-3-(pyrid-2-yl)-1H-pyrazole
To a mixture of 1-methyl-3-(pyrid-2-yl)-1H-pyrazole 876 mg
(5.5 mmol) in concentrated sulphuric acid (25 mL) was carefully
added fuming nitric acid (9 mL). The mixture was stirred at room
temperature for 48 h, then at 100 ꢀC for 1 h. The mixture was then
poured over ice, neutralized using sodium carbonate and extracted
with CHCl3. Purification by chromatography on SiO2 (eluent EtOAc)
gave 470 mg (42%) of a yellow sticky oil. IR (CCl4,
n
¼ cmꢁ1): 2940,
5.3.4.2. 2-Methoxy-N-(1,3-diphenyl-1 H-pyrazol-4-yl) benzamide
3. Yield 64% after crystallization from EtOH. Mp: 202–205 ꢀC. IR
1595, 1545, 1500, 1346. 1H NMR (300 MHz, CDCl3): 3.96 (s, 3H,
NMe), 7.32 (m, 1H, Ar–H), 7.68–7.79 (m, 2H, Ar–H), 8.21 (s, 1H,
]CH–NMe), 8.69 (d, J ¼ 4.5 Hz, 1H, Ar–H). 13C NMR (100 MHz,
CDCl3): 39.9, 123.7, 124.7, 131.3, 132.8, 136.1, 146.1, 148.8, 149.4. MS
m/z: 205 [M þ H]þ, 227 [M þ Na]þ.
(CCl4,
n
¼ cmꢁ1): 3362, 3061,1669,1591,1570,1542,1513,1477,1461,
1446,1389,1295,1259,1233. 1H NMR (300 MHz, CDCl3): 3.64 (s, 3H,
OMe), 6.90 (d, J ¼ 8.6 Hz, 2H, Ar–H), 7.09 (t, J ¼ 7.3 Hz, 1H, Ar–H),
7.18–7.32 (m, 8H, Ar–H), 7.39–7.47 (m, 2H, Ar–H), 8.30 (t, J ¼ 8.1 Hz,
1H, Ar–H), 8.62 (s, 1H, ]CH–N), 9.88 (s, 1H, NH). 13C NMR
(100 MHz, CDCl3): 55.6, 119.25, 120.8, 121.5, 122.0, 124.3, 126.9,
128.7, 128.7, 129.1, 129.3, 129.7, 132.4, 133.2, 134.2, 139.7, 157.1, 162.3.
(MS ESIꢁ): 368 [M ꢁ H]ꢁ. Anal. Calcd for C23H19N3O2: C, 74.78; H,
5.18; N, 11.37. Found: C, 74.71; H, 5.16; N, 11.34.
5.3.3. 4-Amino-1-methyl-3-(pyrid-2-yl)-1H-pyrazole 9
A
mixture of 1-Methyl-4-nitro-3-(pyrid-2-yl)-1H-pyrazole
469 mg (2.3 mmol) and 10% Pd/C (50 mg) in MeOH–DMF (5:1,
15 mL), was shaken under an atmosphere of hydrogen for 5 h. The
mixture was filtered through a plug of celite, the solvent was
evaporated under vacuum to give the title compound (300 mg,
5.3.4.3. N-(1,3-Diphenyl-1H-pyrazol-4-yl)dodecanamide
4. Yield
75%) as a red oil. IR (CCl4,
n
¼ cmꢁ1): 3434, 3331, 3050, 2926, 2283,
81% after crystallization from EtOAc. Mp. 135–137 ꢀC. IR (CCl4,
1550, 1456, 1363, 1254, 1212. 1H NMR (300 MHz, CDCl3): 3.81 (s, 3H,
n
¼ cmꢁ1): 1569.1, 1660.7, 2850.5, 2920.0, 3030.2, 3261.6. 1H NMR
NMe), 4.44 (s, 2H, NH2), 6.95 (s, 1H, ]CH–NMe), 7.06 (dd, J ¼ 8.0,
(400 MHz, CDCl3): 0.88 (t, J ¼ 7.5 Hz, 3H, CH3), 1.20–1.36 (m, 18H,

2030
M. Comes Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 2024–2033
CH2), 2.29 (t, J ¼ 7.6 Hz, 2H, CH2), 6.85 (s,1H, NH), 7.20–7.42 (m,10H,
Ar–H), 8.35 (s, 1H, pyr-H) .13C NMR (100 MHz, CDCl3): 14.1, 22.6,
25.6, 25,7, 28.6, 29.2, 29.2, 29.3, 29.4, 31.8, 36.9, 120.2, 124.5, 127.2,
128.8, 128.9, 129.2, 129.5, 131.7, 134.9, 139.7, 171.1. MS: 418 [M þ H]þ,
440 [M þ Na]þ. Anal. Calcd for C27H35N3O: C, 77.66; H, 8.45; N,
10.06. Found: C, 77.62; H, 8.41; N, 10.02.
5.3.6.1. (S)-2-Amino-N-(1,3-diphenyl-1H-pyrazol-4-yl)propanamide 5
. Starting from commercial N-(tert-butoxycarbonyl)- -alanine,
L
the (S)-2-(tert-butoxycarbonylamino)-N-(1,3-diphenyl-1H-pyrazol-
4-yl)propanamide was obtained in 77% yield as yellow sticky oil
after chromatography on silica (petroleum ether/EtOAc 1/1) 1H NMR
(300 MHz, CDCl3): 1.37 (s, 9H, Boc), 1.40 (d, J ¼ 7.5 Hz, 3H, CH–CH3),
4.23 (q, J ¼ 8.3 Hz, 1H, CH), 4.89 (s, 1H, NHBoc), 7.17–7.30 (m, 8H, Ar–
H), 7.36–7.41 (m, 2H, Ar–H), 8.04 (s, 1H, ]C–NH), 8.34 (s, 1H, pyr-H).
13C NMR (100 MHz, CDCl3): 17.6, 28.5, 50.2, 80.7, 120.4, 124.8, 127.4,
128.8, 129.2, 129.8, 129.4, 129.7, 131.7, 134.2, 139.3, 156.3, 170.6. MS
5.3.4.4. N-[1-Methyl-3-(pyridin-2-yl)-1H-pyrazol-4-yl]benzamide 10
. Yield 79% after crystallization from DCM/n-pentane 1/9. Mp.
119–124 ꢀC. IR (CCl4,
n
¼ cmꢁ1): 3736, 3302, 3071.7, 2938, 2835,
1655, 1554, 1482, 1404, 1358, 1297, 1243.9. 1H NMR (300 MHz,
CDCl3): 3.96 (s, 3H, NMe), 7.03–7.21 (m, 3H, Ar–H), 7.49 (dd,
J ¼ 8.7 Hz, J ¼ 7.5 Hz, 1H, Ar–H), 7.74 (t, J ¼ 7.8 Hz, 1H, Ar–H), 8.07
(d, J ¼ 9.6 Hz, 1H, Ar–H), 8.25–8.40 (m, 2H, Ar–H), 8.51 (s, 1H,
]CH–N), 8.62 (d, J ¼ 5.0 Hz, 1H, Ar–H) 12.03 (s, 1H, NH). 13C NMR
(100 MHz, CDCl3): 39.5, 76.7, 111.4, 119.0, 121.4, 121.6, 121.8, 122.2,
124.9, 132.5 (2C), 132.7, 136.8, 147.6, 154.1, 157.5, 162.5. MS m/z:
279 [M þ H]þ, 301 [M þ Na]þ, 317 [M þ K]þ. Anal. Calcd for
C16H14N4O: C, 69.05; H, 5.07; N, 20.13. Found: C, 69.01; H, 5.03;
N, 20.11.
m/z: 407 [M þ H]þ, 429 [M þ Na]þ. [
a
]D ¼ ꢁ18.7ꢀ (0.39, CHCl3). The
Boc deprotection was performed following the general procedure.
Yield 85%. Mp.112–114 ꢀC. IR (CCl4,
n
¼ cmꢁ1): 3819, 3725, 3652,
3337, 2352, 1683, 1553, 1495. 1H NMR (300 MHz, CDCl3): 1.40 (d,
J ¼ 7.3 Hz, 3H, CH–CH3), 1.60 (s, 2H, NH2), 3.60 (q, J ¼ 7.2 Hz, 1H, CH),
7.19–7.31 (m, 8H, Ar–H), 7.35–7.42 (m, 2H, Ar–H), 8.40 (s, 1H, pyr-H),
9.32 (s, 1H, ]C–NH). 13C NMR (100 MHz, CDCl3): 21.8, 50.9, 120.7,
124.8, 127.3, 128.9, 129.1, 129.6, 129.3, 129.6, 131.5, 134.4, 140.0,
173.1. MS m/z: ESIþ ¼ 307 [M þ H]þ, 329 [M þ Na]þ MS m/z:
ESIꢁ ¼ 305 [M ꢁ H]ꢁ. [
a
]D ¼ þ25ꢀ (0.255, CHCl3). Anal. Calcd for
C18H18N4O: C, 70.57; H, 5.92; N, 18.29. Found: C, 70.52; H, 5.90; N,
18.26.
5.3.4.5. 2-Methoxy-N-(1-methyl-3-pyrid-2-yl-1-H-pyrazol-4-yl) ben-
zamide 11. Yield 73% after crystallization from DCM/n-pentane 1/9.
Mp. 115–116 ꢀC. IR (CCl4,
n
¼ cmꢁ1): 3735, 3300, 3072, 3009, 2936,
5.3.6.2. N-(1,3-Diphenyl-1H-pyrazol-4-yl)-11-mercaptoundecanamide
6. Yield 53%, obtained as solid after chromatography on SiO2
2833, 1653, 1550, 1482, 1404, 1358, 1295. 1H NMR (300 MHz,
CDCl3): 3.96 (s, 3H, NMe), 4.11 (s, 3H, OMe), 7.03–7.21 (m, 3H, Ar–
H), 7.49 (dd, J ¼ 8.7 Hz, J ¼ 7.5 Hz, 1H, Ar–H), 7.74 (t, J ¼ 7.8 Hz, 1H,
Ar–H), 8.07 (d, J ¼ 9.6 Hz, 1H, Ar–H), 8.23 (d, J ¼ 7.8 Hz, 1H, Ar–H),
8.51 (s, 1H, ]CH–N), 8.62 (d, J ¼ 5.0 Hz, 1H, Ar–H) 12.03 (s, 1H, NH).
13C NMR (100 MHz, CDCl3): 39.5, 55.9, 76.7, 111.3, 120.1, 121.1, 121.6,
121.9, 122.8, 124.9, 132.2, 132.7, 136.7, 147.8, 153.9, 157.5, 162.5. MS
m/z: 309 [M þ H]þ, 331 [M þ Na]þ, 347 [M þ K]þ. Anal. Calcd for
C17H16N4O2: C, 66.22; H, 5.23; N, 18.17. Found: C, 66.20; H, 5.20; N,
18.14.
(hexane/EtOAc,1/1). Mp.106–107 ꢀC. IR (CCl4,
n
¼ cmꢁ1): 3434, 2926,
2843, 1695, 1596, 1498, 1446, 1394, 1088. 1H NMR (400 MHz, CDCl3):
0.96–1.40 (m, 13H), 1.54–1.73 (m, 4H), 2.29 (t, J ¼ 7.5 Hz, 2H, –CH2–
CO), 2.52 (q, 2H, J ¼ 7.5 Hz, CH2–SH), 6.87 (s, 1H, NH), 7.15–7.33 (m,
8H, Ar–H), 7.34–7.44 (m, 2H, Ar–H), 8.31 (s, 1H, ]CH–N). 13C NMR
(100 MHz, CDCl3): 24.6, 25.63, 28.4, 29.2 (2C), 29.3, 29.5, 29.6, 34.0,
36.8, 120.5, 124.5, 127.1, 128.8, 128.9, 129.2, 129.5, 131.9, 132.3, 134.9,
139.9, 171.1. MS m/z: 436 [M þ H]þ, 458 [M þ Na]þ. Anal. Calcd for
C26H33N3OS: C, 71.69; H, 7.64; N, 9.65. Found: C, 71.66; H, 7.60; N,
9.61.
5.3.4.6. N-(1-Methyl-3-(pyridin-2-yl)-1H-pyrazol-4-yl)dodecanamide
12. Yield 65% after crystallization from pentane. Mp. 59–60 ꢀC. IR
5.3.6.3. 12-Amino-N-(1,3-diphenyl-1H-pyrazol-4-yl)dodecanamide 7.
12-(tert-butoxycarbonylamino)dodecanoic acid was obtained
according to Ref. [22]. Starting from this protected amino
acid the 12-N-(tert-butoxycarbonyl)amino-N-(1,3-diphenyl-1H-
pyrazol-4-yl)dodecanamide 17 was obtained in 89% yield after
chromatography on SiO2 (hexane/EtOAc, 1/1). Mp.116–118 ꢀC. 1H
NMR (300 MHz, CDCl3): 1.18–1.32 (m, 16H, –CH2–), 1.38–1.42 (s, 9H,
–CH3), 1.60 (quint, J ¼ 7.2 Hz, 2H, –CH2–CH2–CO), 2.24 (t, J ¼ 7.2 Hz,
2H, –CH2–CO), 3.04 (q, J ¼ 6.0 Hz, 2H, –CH2–NH), 4.60 (s, 1H, NH),
7.13–7.24 (m, 8H, Ar–H), 7.32–7.37 (m, 2H, Ar–H), 8.19 (s, 1H, ]CH–
N). 13C NMR (75.3 MHz, CDCl3): 24.5, 25.3, 25.5, 26.6, 28.3, 29.1,
29.2, 29.3, 29.3, 29.9, 34.7, 36.6, 40.5, 55.6, 78.7, 120.2, 124.4, 127.1,
128.6, 128.9, 129.4, 132.1, 135.1, 139.6, 155.8, 171.4. MS (m/z): 533
[M þ H]þ. Boc removal was done according to the general procedure
to yield 85% of product 7.
(CCl4,
n
¼ cmꢁ1): 3300, 2916, 2853, 1674, 1592, 1550, 1488, 1457,
1405, 1363, 1332, 1267, 1228. 1H NMR (400 Hz, CDCl3): 0.85 (t,
J ¼ 7.0 Hz, 3H, CH2–CH3), 1.18–1.43 (m, 16H, –CH2–), 1.75–1.80 (m,
2H), 2.42 (t, J ¼ 7.5 Hz, 2H, CH2–CO), 3.90 (s, 3H, NMe), 7.14 (t,
J ¼ 6.2 Hz, 1H, Ar–H), 7.71 (t, J ¼ 8.3 Hz, 1H, Ar–H), 8.01 (d, J ¼ 8.3 Hz,
1H, Ar–H), 8.22 (s, 1H, ]CH–N), 8.52 (d, J ¼ 5.0 Hz, 1H, Ar–H), 10.85
(s, 1H, NH) .13C NMR (100 MHz, CDCl3): 14.0, 22.6, 25.6, 25.9, 29.2
(2C), 29.4 (2C), 31.8, 37.1, 39.4, 119.5, 121.5, 122.0, 123.0, 136.6, 136.8,
147.9, 153.9, 170.4 (NC]O). MS: 357 [M þ H]þ, 379 [M þ Na]þ. Anal.
Calcd for C21H32N4O: C, 70.75; H, 9.05; N, 15.72. Found: C, 70.72; H,
9.02; N, 15.70.
5.3.5. General procedure for the Boc deprotection
The Boc deprotection was performed starting from 0.5 mmol of
product dissolved in EtOAc (5 mL) and adding 0.5 mL of 3 M HCl in
EtOAc. After 2–8 h at room temperature (following by TLC), the
solvent was evaporated to yield pure product as chlorohydrate,
then the free amine was obtained after basification with conc.
ammonia and extraction with EtOAc.
5.3.6.4. 12-Amino-N-(1,3-diphenyl-1H-pyrazol-4-yl)dodecanamide 7.
Mp. 115–118 ꢀC. IR (CCl4,
n
¼ cmꢁ1): 3838, 3734, 2925, 2852, 1647,
1558, 1534. 1H NMR (300 MHz, CDCl3): 1.21–1.37 (m, 14H, –CH2–),
1.43 (quint, 2H, –CH2–CH2–NH2), 1.67 (quint, 2H, –CH2–CH2–CO),
1.77 (s, 2H, NH2), 2.28 (t, J ¼ 7.5 Hz, 2H, –CH2–CO), 2.67 (t, J ¼ 6.9 Hz,
2H, CH2–NH2), 7.00 (s, 1H, NH), 7.17–7.31 (m, 8H, Ar–H), 7.36–7.42
(m, 2H, Ar–H), 8.29 (s, 1H, ]CH–N). 13C NMR (100 MHz, CDCl3):
25.1, 25.3, 25.9, 26.7, 27.2, 29.5, 29.6, 29.7, 29.8, 31.2, 33.1, 33.7,
34.3, 37.2, 42.4, 76.9, 120.7, 124.9, 127.5, 129.1, 129.2, 129.5,
129.9, 132.2, 135.34, 140.1, 171.6 (Extra carbon signals are due to
rotamers). MS m/z: 433 [M þ H]þ, 455 [M þ Na]þ. Anal. Calcd for
C27H36N4O: C, 74.96; H, 8.39; N, 12.95. Found: C, 74.93; H, 8.36; N,
12.91.
5.3.6. General procedure for the synthesis of compounds 5, 6, 7, 8
and 13, 14, 15, 16
To a stirred mixture of the appropriate acid (1 mmol) 4-dime-
thylaminopyridine (DMAP, 0.2 mmol) in DCM (10 mL), product 1 or
9 (1 mmol) and dicyclohexylcarbodiimide (DCC, 1.2 mmol) were
added at 0 ꢀC. The mixture was stirred 24 h at room temperature
then was filtered, dried, concentrated and purified by column
chromatography on SiO2, using an EtOAc gradient.

M. Comes Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 2024–2033
2031
5.3.6.5. N-(1,3-Diphenyl-1H-pyrazol-4-yl)-11-hydroxyundecanamide
8. Yield 41% obtained as solid after chromatography on SiO2
Boc deprotection was done according to the general procedure
affording 43% of product 15 as a white solid. Mp. 63–68 ꢀC. IR (CCl4,
(hexane/EtOAc, 1.5/1). Mp. 100–102 ꢀC. IR (CCl4,
n
¼ cmꢁ1): 3430,
n
¼ cmꢁ1): 3301, 2961, 2843, 1674,1591, 1545,1488, 1456,1404, 1358,
3059, 2914, 2852, 1600, 1502, 1445, 1393. 1H NMR (400 MHz,
CDCl3): 1.10–1.30 (m, 14H), 1.40–1.85 (m, 4H,), 2.30 (t, 2H,
J ¼ 7.4 Hz), 3.52 (q, 2H, J ¼ 7.1 Hz), 6.90 (s,1H, NH), 7.20–7.35 (m, 8H,
Ar–H), 7.55–7.65 (m, 2H, Ar–H), 8.20 (s, 1H, ]CH–N). 13C NMR
(100 MHz, CDCl3): 24.4, 25.9, 28.8, 29.2–29.8 (5C), 34.4, 36.1, 121.1,
124.9, 127.9, 128.8–129.9 (4C),131.0,135.4,138.3,170.0. MS m/z: 420
[M þ H]þ, 442 [M þ Na]þ. Anal. Calcd for C27H35N3O2: C, 74.79; H,
8.14; N, 9.69. Found: C, 74.72; H, 8.11; N, 9.66.
1269, 1145, 1093. 1H NMR (300 MHz, CDCl3): 1.07–1.37 (m, 16H),
1.62–1.80 (m, 2H, –CH2–CH2–CO) 2.30 (t, J ¼ 7.3 Hz, 2H, –CH2–CO)
2.55 (t, J ¼ 6.9 Hz, 2H, –CH2–NH) 3.25 (s, 2H, NH2), 3.79 (s, 3H, NMe),
7.02 (t, J ¼ 6.3 Hz, 1H, Ar–H), 7.57 (t, J ¼ 7.5 Hz, 1H, Ar–H), 7.88 (d,
J ¼ 7.8 Hz, 1H, Ar–H), 8.10 (s, 1H, ]CH–NMe), 8.39 (d, J ¼ 4.8 Hz, 1H,
Ar–H), 10.72 (s, 1H, NH). 13C NMR (100 MHz, CDCl3): 25.3, 26.5, 28.9,
29.0 (3 C), 29.2 (2 C), 32.2, 36.7, 39.1 (Me), 41.2, 119.1, 121.2, 121.7,
122.5, 136.3, 136.4, 147.6, 153.6, 169.9. MS m/z: 372 [M þ H]þ, 394
[M þ Na]þ. Anal. Calcd for C21H33N5O: C, 67.89; H, 8.95; N, 18.85.
Found: C, 67.86; H, 8.92; N, 18.82.
5.3.6.6. (2S)-2-Amino-N-[1-methyl-3-(pyrid-2-yl)-1H-pyrazol-4-yl]-
propanamide 13. Starting from commercial N-(tert-butoxycarbonyl)-
L-alanine, the (2S)-2-(tert-butoxycarbonylamino)-N-[1-methyl-3-
5.3.6.9.
11-Hydroxy-N-[1-methyl-3-(pyrid-2-yl)-1H-pyrazol-4-yl]-
(pyrid-2-yl)-1H-pyrazol-4-yl]propanamidewasobtained in70%yield
after chromatography on SiO2 (petroleum ether/EtOAc 1/1) as
undecanamide 16. Yield 44% obtained as solid after chromatography
on SiO2 (hexane/EtOAc, 3/1). Mp: 74–77 ꢀC. IR (CCl4,
n
¼ cmꢁ1): 3300,
a yellow sticky oil. IR (CCl4,
n
¼ cmꢁ1): 3434, 3269, 2968, 2926, 2283,
3105, 2921, 2853, 1675, 1590, 1550, 1451, 1404, 1269, 1088. 1H NMR
(400 MHz, CDCl3): 1.15–1.30 (m, 14H), 1.30–1.45 (m, 5H), 2.25 (t,
J ¼ 7.6 Hz, 2H), 3.60 (t, J ¼ 7.6 Hz, 2H), 3.90 (s, 3H, NMe), 7.15–7.20 (m,
1H, Ar–H), 7.65–7.70 (m,1H, Ar–H), 7.92 (d, J ¼ 8.1 Hz,1H, Ar–H), 8.23
(s,1H, ]CH–NMe), 8.55 (d, J ¼ 5.0 Hz,1H, Ar–H),10.70 (s,1H, NH).13C
NMR (100 MHz, CDCl3): 24.2, 25.9, 28.2, 29.2–29.8 (5C), 34.1, 38.3,
40.1,120.4,121.2,122.6,123.4,137.4,137.8,148.0,153.3,169.7. MS m/z:
359 [M þ H]þ, 381 [M þ Na]þ. Anal. Calcd for C21H32N4O2: C, 67.71; H,
8.66; N, 15.04. Found: C, 67.70; H, 8.62; N, 15.02.
1996, 18416, 1716, 1674, 1544, 1401, 1363, 1294, 1213. 1H NMR
(400 MHz, CDCl3): 0.79–1.50 (m, 12H), 3.87 (s, 3H, NMe), 4.41 (s, 1H,
N–H), 5.36 (q, J ¼ 7.6 Hz, 1H, CH–CH3), 7.12 (t, J ¼ 6.6 Hz, 1H, Ar–H),
7.67 (t, J ¼ 6.6 Hz, 1H, Ar–H), 7.96 (d, J ¼ 8.4 Hz, 1H, Ar–H), 8.16 (s, 1H,
]CH–N), 8.52 (d, J ¼ 4.2 Hz, 1H, Ar–H), 11.40 (s, 1H, NH). 13C NMR
(100 MHz, CDCl3): 18.8, 28.2 (3C), 39.3, 50.5, 76.7, 79.7, 119.3, 121.4,
121.5, 122.8, 136.7, 136.9, 147.8, 153.5, 155.1, 169.9. MS m/z: 368
[M þ Na]þ 384 [M þ K]þ. The Boc deprotection was performed
according to the general procedure yielding 13 in 75%. Mp: 105–
108 ꢀC from DCM/pentane. IR (CCl4,
n
¼ cmꢁ1): 3259, 3051, 2957,
5.3.7. General procedure for the synthesis of the thioamide
According to: Ref. [23].
2926, 1669, 1591, 1550, 1482, 1451, 1409, 1358, 1327, 1275. 1H NMR
(300 MHz, CDCl3): 1.44 (d, J ¼ 7.1 Hz, 3H, CH–CH3), 1.90 (s, 2H, NH2),
3.67 (q, J ¼ 6.7 Hz,1H, CH–CH3), 3.91 (s, 3H, NMe), 7.15 (dd, J1 ¼7.8 Hz,
J2 ¼ 5.0 Hz,1H, Ar–H), 7.71 (t, J ¼ 8.1 Hz,1H, Ar–H), 8.00 (d, J ¼ 8.1 Hz,
1H, Ar–H), 8.26 (s,1H, ]CH–N), 8.55 (d, J ¼ 5.0 Hz,1H, Ar–H),11.66 (s,
1H, NH).13CNMR (100 MHz, CDCl3): 21.5, 39.2, 50.9,119.2,121.3,122.7,
136.5,137.3,148.0,153.5,173.3. MS m/z: 246 [M þ H]þ, 268 [M þ Na]þ.
12-(tert-butoxycarbonylamino)-N-[1,3-diphenyl-1H-pyrazol-4-
yl]dodecanamide 17 (1 mmol) and Lawesson Reagent (1 mmol) in
dry THF (20 mL) were refluxed until the starting amide had been
consumed (TLC, hexane/EtOAc, 1/1, about 24 h). Purification by
silica gel chromatography with hexane/EtOAc 1/1 afforded the 18.
[
a
]D ¼ þ7.51ꢀ (0.415, CHCl3). Anal. Calcd for C12H15N5O: C, 58.76; H,
5.3.7.1. tert-Butyl 12-(1,3-diphenyl-1H-pyrazol-4-yl-amino)-12-thi-
6.16; N, 28.55. Found: C, 58.72; H, 6.13; N, 28.54.
oxododecylcarbamate 18. 95% yield as yellow sticky oil. IR (CCl4,
n
¼ cmꢁ1): 3452, 3366, 2916, 2844, 1720, 1500, 1389, 1243, 1169. 1H
5.3.6.7. 11-Mercapto-N-(1-methyl-3-(pyridin-2-yl)-1H-pyrazol-4-yl)-
undecanamide 14. Yield 53% obtained as solid. Mp: 78–79 ꢀC. IR
NMR (400 MHz, CDCl3): 1.05–1.15 (m, 14H), 1.40 (bs, 9H), 1.65–1.70
(m, 4H), 2.65 (t, J ¼ 7.7 Hz, 2H), (at 2.45 resonates a triplet corre-
sponding to the minor stereoisomer), 3.00 (bs, 2H), 4.60 (bs, 1H),
7.05–7.40 (m,10H, Ar–H), 8.35 (s,1H, CH]), 8.85 (bs,1H, NHCO). 13C
NMR (100 MHz, CDCl3): 26.6, 28.2, 28.6, 29.06, 29.13, 29.21, 29.28,
29.41, 29.85. 40.4, 47.3, 79.0, 121.6, 124.6, 127.3, 128.7, 128.8, 129.0,
129.1, 129.3, 135.5, 136.5, 139.2, 156.0, 205.9. MS m/z: 549 [M þ H]þ,
571 [M þ Na]þ.
(CCl4,
n
¼ cmꢁ1): 3300, 2923, 2849, 1675, 1591, 1544, 1450, 1400,
1256, 1088. 1H NMR (300 MHz, CDCl3): 1.06–1.60 (m, 17H), 2. 24
(t, J ¼ 7.0 Hz, 2H, CH2), 2.49 (q, J ¼ 8.2 Hz, 2H, CH2), 3.86 (s, 3H, NMe),
7.11 (bt, J ¼ 4.9 Hz, 1H, Ar–H), 7.75 (td, J ¼ 7.4 Hz, J ¼ 1.7 Hz, 1H,
Ar–H), 8.02 (d, J ¼ 9.2 Hz, 1H, Ar–H), 8.19 (s, 1H, Ar–H), 8.55
(bd, J ¼ 6.1 Hz, Ar–H), 10.8 (s, 1H, NH, Ar–H). 13C NMR (100 MHz,
CDCl3): 24.9, 25.9, 28.7, 29.6, 29.7, 29.8 (2 C), 29.9, 34.4, 37.5, 39.7,
119.8,121.8, 122.4, 123.3, 136.9, 137.2, 148.3,154.3, 170.7. MS m/z: 397
[M þ Na]þ. Anal. Calcd for C20H30N4OS: C, 64.14; H, 8.07; N, 14.96.
Found: C, 64.13; H, 8.04; N, 14.92.
5.3.7.2. 12-amino-N-(1,3-diphenyl-1H-pyrazol-4-yl)dodecanethioa-
mide 19. 12-Amino-N-(1,3-diphenyl-1H-pyrazol-4-yl)dodecane-
thioamide 19: Mp: 99–102 ꢀC. IR (CCl4,
n
¼ cmꢁ1): 3402, 3173, 2924,
2841, 1597, 1497, 1447, 1392, 1068. 1H NMR (400 MHz, CDCl3): 1.05–
1.35 (m, 12H), 1.80–1.95 (m, 6H), 1.85–1.95 (m, 2H), 2.70 (t,
J ¼ 7.7 Hz, 2H), (at 2.60 resonates a triplet corresponding to the
minor rotamer), 2.90 (bs, 2H), 7.10–7.40 (m, 10H, Ar–H), 8.25 (bs,
1H, NH), 8.45 (s, 1H, CH]). 13C NMR (100 MHz, CDCl3): 26.4, 27.6,
28.7, 28.9, 29.2 (2C), 29.3 (2C), 29.5, 39.9, 47.7, 121.8, 124.7, 127.5,
128.0, 128.8, 129.1, 129.2, 129.5, 135.2, 136.3, 139.4, 205.3. MS m/z:
449 (M þ 1), 471 (M þ Na). Anal. Calcd for C27H36N4S: C, 72.28; H,
8.09; N, 12.49. Found: C, 72.26; H, 8.07; N, 12.47. Boc removal was
accomplished according to the general procedure giving in 95%
yield a pale yellow solid which was crystallised from Et2O.
5.3.6.8. 12-Amino-N-[1-methyl-3-(pyrid-2-yl)-1H-pyrazol-4-yl]dode-
canamide 15. Starting from N-Boc-protected-12-aminododecanoic
acid the 12-(tert-butoxycarbonylamino)-N-[1-methyl-3-(pyrid-2-
yl)-1H-pyrazol-4-yl]dodecanamide was obtained in 62% yield after
chromatography on SiO2 (hexane/EtOAc 3/1) as a white solid. Mp:
60–62 ꢀC. 1H NMR (300 MHz, CDCl3): 1.23–1.47 (m, 21H), 1.57 (m,
4H) 1.76–1.85 (m, 2H, –CH2–CH2–CO), 2.44 (t, J ¼ 7.7 Hz, 2H, –CH2–
CO), 3.09 (q, J ¼ 7.3 Hz, 2H, –CH2–NH), 3.92 (s, 3H, NMe), 4.49 (s, 1H,
NH), 7.18 (dd, J1 ¼7.5 Hz, J2 ¼ 5.1 Hz, 1H, Ar–H), 7.74 (dd, J1 ¼8.6 Hz,
J2 ¼ 7.9 Hz, 1H, Ar–H), 8.03 (d, J ¼ 8.2 Hz, 1H, Ar–H), 8.24 (s, 1H,
]CH–N), 8.54 (d, J ¼ 5.0 Hz, 1H, Ar–H), 10.86 (s, 1H, NH). 13C NMR
(75.3 MHz, CDCl3): 25.1, 26.3, 27.9 (3C, Boc), 28.7, 28.8 (2C), 28.9, 29.0
(2C), 29.6, 36.6, 38.9, 40.1, 78.1, 119.3, 121.1, 121.6, 122.5, 136.1, 136.3,
147.4,153.5,155.6,169.7. MS (m/z): 472 [M þ H]þ, 494 [M þ Na]þ. The
5.3.8. Synthesis of PLGA-b-PEG
PLGA–COOH (3.0 g, 0.43 mmol) and N-hydroxysuccinimide
(NHS, 195 mg, 1.7 mmol) were dissolved in anhydrous methylene

2032
M. Comes Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 2024–2033
chloride (15 mL). After cooling at 0 ꢀC N,N0-dicyclohexyl-
carbodiimide (DCC, 380 mg, 1.8 mmol) was added and the mixture
was left to warm up at room temperature and react for 20 h.
Afterwards, dicyclohexylurea (DCU) was removed by filtration and
PLGA–NHS was precipitated with cold ethyl ether (20 mL), and
repeatedly washed with the same solvent (3 ꢂ 10 mL). After drying
under vacuum, the resulted white solid of PLGA–NHS (2.7 g) was
collected, stored at ꢁ20 ꢀC for no more than a week and used in the
following step without further purification. 1H NMR (300 MHz;
dissolved in 100 ml of DMSO per well. The spectophotometric
absorbance was then measured at 540 nm using a BIO-RAD
Microplate Reader Benchmark (Software Microplate Manager 5.1).
The same method was used to test the cytotoxicity of pyrazoles
encapsulated in PEGylated polymeric NPs. Control samples were
incubated with NPs at 24, 48 and 72 h.
Acknowledgment
CDCl3)
d 5.11–5.31 (m, 4.63–4.92 m), 2.8 (s, 1.50–1.61 m).
This work has been supported by University of Bologna (Funds
for Selected Research Topics). We thank Prof. A. Ricci, Department
of Organic Chemistry for helpful discussions.
PLGA–NHS (1.8 g, 0.25 mmol) was dissolved in anhydrous chloro-
form (10 mL) followed by addition of PEG–NH2 (540 mg,
0.27 mmol) and N,N-diisopropylethylamine (0.125 mL, 0.72 mmol).
After 18 h the co-polymer was precipitated with cold ethyl ether
(20 mL) and washed with the same solvent (3 ꢂ10 mL) and cold
water (3 ꢂ 10 mL) to remove unreacted PEG–NH2. The resulting
white solid of PLGA-b-PEG block co-polymer (1.6 g) was dried
under vacuum, stored at ꢁ20 ꢀC and used without further treat-
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