Novel valproic aminophenol amides with enhanced glial cell viability effect

Article abstract of DOI:10.1039/c7ra00048k

New valproic acid derivatives were synthesized by coupling valproyl chloride with ortho-aminophenols, resulting in seven N-(ortho-hydroxyphenyl)valproamides. These amides share similar structural characteristics and exhibit tuneable electronic and steric contributions either without particular substituents, or through the inclusion of electro-donating (-Me), electro-withdrawing (-NO₂) or pi electro-donating/sigma electro-withdrawing (-Cl) substituents at the aromatic ring. The identity of such derivatives was evidenced through spectroscopic characterization using FTIR, ¹H, ¹³C and HETCOR NMR, as well as by analyzing their melting points. In particular, for three derivatives it was feasible to determine their chemical structures in the crystal phase; all three behaved in a similar fashion and exhibited very similar conformations independent of the attached substituents. The base compound was found to exhibit 15.8 times more activity in C6 cells and 4.4 times more activity in U373 cells compared with VPA. In general, the parent compound, or those having-Me as a substituent, presented a greater effect on C6 cells than U373 cells. However, those with-NO₂ and-Cl substituents, as well as VPA, required similar doses for the IC₅₀ in both cell lines. Modification of the base compound with a-Me or-NO₂ substituent increased the effect on cell viability to ca. 20 times that of VPA in both C6 and U373, indicating that a larger structure causes an important enhancement in the inhibition of cell viability. In both cell lines,-Cl containing derivatives were the most active compounds. For these derivatives, an activity increase of ca. 59 and 47 times that of VPA was observed for C6 and U373 cells, respectively. An important perspective is that VPA analogues possessing an aromatic ring with a-Cl substituent may become central structures in the search for more potent pharmaceutical prototypes.

Full text of DOI:10.1039/c7ra00048k

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PAPER
Novel valproic aminophenol amides with enhanced
glial cell viability effect†
Cite this: RSC Adv., 2017, 7, 12391
Andrea Alpuche-Garcıa, Xochitl Davila-Gonzalez, Leticia Arregui*
´
´
´
and Hiram I. Beltran*
´
New valproic acid derivatives were synthesized by coupling valproyl chloride with ortho-aminophenols,
resulting in seven N-(ortho-hydroxyphenyl)valproamides. These amides share similar structural
characteristics and exhibit tuneable electronic and steric contributions either without particular
substituents, or through the inclusion of electro-donating (–Me), electro-withdrawing (–NO
2
) or pi
electro-donating/sigma electro-withdrawing (–Cl) substituents at the aromatic ring. The identity of such
1
13
derivatives was evidenced through spectroscopic characterization using FTIR, H, C and HETCOR NMR,
as well as by analyzing their melting points. In particular, for three derivatives it was feasible to determine
their chemical structures in the crystal phase; all three behaved in a similar fashion and exhibited very
similar conformations independent of the attached substituents. The base compound was found to
exhibit 15.8 times more activity in C6 cells and 4.4 times more activity in U373 cells compared with VPA.
In general, the parent compound, or those having –Me as a substituent, presented a greater effect on
C6 cells than U373 cells. However, those with –NO
2
and –Cl substituents, as well as VPA, required
similar doses for the IC50 in both cell lines. Modification of the base compound with a –Me or –NO
2
substituent increased the effect on cell viability to ca. 20 times that of VPA in both C6 and U373,
indicating that a larger structure causes an important enhancement in the inhibition of cell viability. In
both cell lines, –Cl containing derivatives were the most active compounds. For these derivatives, an
activity increase of ca. 59 and 47 times that of VPA was observed for C6 and U373 cells, respectively. An
important perspective is that VPA analogues possessing an aromatic ring with a –Cl substituent may
become central structures in the search for more potent pharmaceutical prototypes.
Received 3rd January 2017
Accepted 23rd January 2017
DOI: 10.1039/c7ra00048k
rsc.li/rsc-advances
progression. VPA and other iHDAC have been explored for
glioblastoma treatment alone or with chemotherapy or radio-
Introduction
Valproic acid (VPA) is a fatty acid with anticonvulsant properties therapy, and promising effects have been found. However,
used in the treatment of epilepsy, and this drug also has anti- additional efforts to nd more effective drugs are required, in
tumor activity due to its effects as a histone deacetylase inhib- particular those involving the testing of drugs in glioblastoma
1
8
itor. The occurrence of many types of cancer are generally cell lines, given the previously reported evidence.
2
accompanied by histone hypoacetylation. Because of this,
In order to diminish or avoid the side effects associated
many new strategies to ght cancer are focused on the resto- with VPA, as well as to enhance its activity, a common and
ration of de-acetylation balance through the use of histone useful strategy has been the chemical modication of the
3–6
9
deacetylase inhibitors (iHDAC) such as VPA. Nevertheless, parent compound. For instance, in 2005, Eyal et al. evaluated
despite the promising effects offered by VPA against cancer, it and compared the iHDAC of VPA and its constituent isomers,
causes side effects mainly related to blood and bone marrow including valnoctic acid (VCA), propylisopropylacetic acid
toxicity, and high concentrations are required to achieve the (PIA), diisopropylacetic acid (DIA), 2,2,3,3-tetramethylcyclo-
7
desired pharmacological activity, mostly in the mM range.
propylcarboxylic acid (TMCA), and VPA metabolites: 2-en-AVP
Glioblastoma is the most common and devastating primary and 4-en-AVP. The authors found that almost any chemical
tumour in adults. Epigenetic changes together with genetic modication of the VPA structure results in the decay of its
modications have been associated with its generation and activity as iHDAC. Another strategy, developed in 2006 by
1
0
Deubzer and co-workers, was the employment of VPA
analogues with longer side chains, as well as the inclusion of
alkenyl fragments in the structure. These changes caused
Departamento de Ciencias Naturales, DCNI, UAM Cuajimalpa, 05300, Ciudad de
M ´e xico, Mexico. E-mail: arregui@correo.cua.uam.mx; hbeltran@correo.cua.uam.mx
†
Electronic supplementary information (ESI) available: CCDC no. 1525302 for multifunctional properties of such derivatives as iHDAC,
2 3 6
VA , 1525303 for VA , and 1525301 for VA . For ESI and crystallographic data in
showing interesting cell cycle modulation due to the induction
CIF or other electronic format see DOI: 10.1039/c7ra00048k
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of p21 expression, as well as low toxicity on CD34+ bone Fourier transform infrared (FT-IR) spectra
1
0
marrow cells. Another recent effort to enhance the benecial
effects of VPA derivatives was carried out using phosphoval-
proic acid, employed as iHDAC, which involved tracking its
dominant molecular target STAT3. This compound includes
ester functionality to anchor the VPA fragment to the diethyl (4-
hydroxybenzyl) phosphate parent compound. This compound
has shown synergistic behaviour in enhanced pancreatic cancer
ꢀ1
The FT-IR spectra were recorded in the range of 4000–400 cm
on a Bruker Tensor-27 FT-IR spectrophotometer, using KBr
pellets or KBr mixtures of each of the isolated samples. The
samples were measured using the ATR technique.
Single crystal X-ray diffraction (X-ray)
11
inhibition, compared with VPA. Lastly, in 2009, the compound X-ray diffraction data were acquired on an Enraf Nonius
˚
N-9-(2-hydroxy)ethoxymethylguanine disubstituted with VPA was diffractometer FR590 Kappa-CCD (lMoKa ¼ 0.71073 A, graphite
1
2
patented and in 2014 it was evaluated by Tarasenko and co- monochromator, T ¼ 293 K, using rotating image scan with
1
3
workers as an anticancer agent in various cell lines with CCD). Monocrystals were mounted in LINDEMAN tubes. Reec-
promising results. In particular, ester and amide groups link this tions were corrected for Lorentz and polarization effects. The
16
compound to the VPA moiety, indicating that these functional- SHELX-S and SHELX-L 2014 program suite was used for the rst
ities should work to generate new promising derivatives of VPA. structural solution, as well as for the renement and output data.
Some other trials to nd potent VPA analogues with enhanced These programs, as well as the molecular perspectives, were
14
17
iHDAC activity have also emerged lately.
employed and obtained in the OLEX2 environment. All heavy
Thus, the current work aims to obtain and characterize seven atoms were found through electronic density differences in the
new VPA derivatives, wherein the molecular design strategy is Fourier maps and rened anisotropically. Some of the hydrogen
not to vary the valproic fragment, but instead the carboxylate atoms were also found through Fourier maps and rened iso-
moiety, through the formation of amidic functionalities. This tropically, and the remnants were geometrically calculated and
change is accompanied by the inclusion of an aromatic ring added for the nal renement.
containing different electro-withdrawing or electro-releasing
15
substituents in order to modulate bioactivities. Finally, the
new structures recover the OH fragment present in VPA,
through the inclusion of a phenolic group. The results of this
contribution are that the seven valproic amides exhibit an
enhanced effect on cell viability compared with VPA in two
glioma cell lines.
Thin layer chromatography (TLC)
Thin layer chromatography (TLC) aluminium sheet plates of
silica 60 F254 of 0.2 mm width (Merck, KGaA) were employed to
track the course of each reaction. The TLC separation of each
compound was developed using a hexane/ethyl acetate 1 : 1
solvent mixture and the plates were revealed using a UV-vis
lamp. In all obtained VA products, the appearance of single
spots was indicative of their purity and also conrmed that the
desired reaction had been achieved.
Experimental section
General information
Available solvents and reagents were used as received. 2-Pro-
pylpentanoic acid (VPA, CAT P6273-100ML), 2-amino-p-cresol
Synthetic methodology for compounds VA1–7
1
Synthesis of VA . In a round ask, 0.5 g (3.5 mmol) of VPA
(APh2, CAT 144908-50G), 2-amino-5-methyl-phenol (APh3, CAT
was poured and dissolved in 40 mL of CH CN. Then, 0.68 mL
3
144916-10G), 2-amino-4-nitro-phenol (APh4, CAT A70402-5G), 2-
(
3.5 mmol) of SOCl was added to yield the acid chloride of VPA
2
amino-5-nitro-phenol (APh5, CAT 303585-25G), 2-amino-4-
chloro-phenol (APh6, CAT C44400-100G) and 2-amino-5-
chloro-phenol (APh7, CAT 552224-25G) were acquired from
Sigma-Aldrich; 2-aminophenol (APh1, CAT 09110-100G) was
obtained from Fluka. The solvents employed were acetonitrile
18
in situ. This stage was carried out for 24 h at room tempera-
ture, keeping the system closed with a glass cap. Then 0.38 g
3
(3.5 mmol) of APh1 was predissolved in 5 mL of CH CN and 5
mL of triethylamine was added to the round ask drop by drop
through an addition funnel, followed by magnetic stirring for
an hour. The mixture was set to reux at a temperature which
was maintained for 24 h to nally yield the N-(2-hydroxyphenyl)-
(CH
3
CN, CAT 9011-03-4L), tetrahydrofuran (THF, CAT 9450-03-
4
9
L), ethyl acetate (AcOEt, CAT 75-05-8-2L) and hexane (Hex, CAT
30L) and were acquired from Reasol.
2
-propylpentanamide (AV ). The completion of the reaction was
1
followed by FTIR carbonyl band displacement of the carboxylic
acid, acyl chloride and nally secondary amide groups, as well
as by thin layer chromatography of the reaction crudes. Vola-
1
D & 2D nuclear magnetic resonance spectroscopy (1D & 2D
NMR)
NMR experiments were performed on a VARIAN Mercury 200-BB tiles were removed under reduced pressure, and 20 mL of THF
1
spectrometer and on an Anasazi spectrometer EFT-60. The H was added to the remaining solids, thus keeping the triethy-
1
3
and C chemical shis [d ¼ ppm] were taken as relative to lammonium chloride salt insoluble, which was separated
1
13
internal SiMe (TMS, d( H) ¼ 0.00 ppm, d( C) ¼ 0.00 ppm), and through ltration. The soluble fraction was dried through
4
the coupling constants were quoted in Hz. The employed reduced pressure distillation and the remnant solid was
solvent was CDCl , and just for the compounds AV4 and AV5, recrystallized in an ethyl acetate/hexane 1 : 4 solvent mixture. In
3
0.1 mL of DMSO-d
6
was added to achieve full dissolution due to this way it was feasible to obtain compound VA
1
in a yield of
the presence of nitro groups.
64.8%. Similar to VA , the other six valproic amides (VA2–7) were
1
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prepared by just varying the corresponding ortho-aminophenol 126.7 (C2, 1C), 120.8 (C5, 1C), 116.8 (C3, 1C), 116.0 (C6, 1C), 47.8
source. As stated in a very recent contribution, this synthetic (C8, 1C), 35.1 (C9, 2C), 20.6 (C10, C2), 14.0 (C11, 2C).
strategy solely provided N-acylation of the carboxylate moiety,
instead of O-acylation.
N-(2-Hydroxy-4-nitrophenyl)-2-propylpentanamide
(VA ).
5
ꢀ1
19
ꢁ
Orange solid, yield 30.6%, mp 161–163 C. FTIR (n ¼ cm
,
N-(2-Hydroxyphenyl)-2-propylpentanamide (VA
1
). Light KBr): 3600–2400 (O–H), 3408 (N–H), 3096 (Csp
2
–H), 2957, 2934,
ꢁ
ꢀ1
brown solid, yield 64.8%, mp 55–57 C. FTIR (n ¼ cm , KBr): 2872 (Csp
3
–H), 2362, 2347, 1669, 1627 (–C(]O)–N–), 1592, 1506
3
600–2600 (O–H), 3256 (N–H), 3077 (Csp
2
–H), 2957, 2929, 2873 (–C(O)]N–), 1465, 1424, 1400, 1346 (–N(]O)–O), 1327, 1268,
1
(
1
C
sp
3
–H), 1626, 1603 (–C(]O)–N–), 1545, 1497 (–C(O)]N–), 1247, 1194, 896, 875, 825, 880, 743. NMR H (d ¼ ppm, CDCl
3
, J
¼ 8.88,
Hz): 8.95, 8.73 (OH, sb, 1H & NH, s, 1H), 7.44 (H3, d, J ¼ 7.25, 1H), 7.76 (H4, d, J ¼ 8.88, 1H), 7.63 (H6, s, 1H), 2.43 (H8, q, J ¼
1
387, 1313, 1264, 772, 750, 722, 693. H NMR (d ¼ ppm, CDCl
3
, J ¼ Hz): 9.07, 8.35 (OH, sb, 1H & NH, s, 1H), 8.20 (H3, d, J
o
¼
o
o
1
H), 7.02 (H5, t, J ¼ 7.25, 1H), 6.95 (H4, t, J ¼ 7.25, 1H), 6.83 7.14, 1H), 1.48 (H9, m, 4H), 1.36 (H2, m, 4H), 0.92 (H11, t, J ¼
o
o
13
(
4
H6, d, J ¼ 7.25, 1H), 2.46 (H8, q, J ¼ 6.42, 1H), 1.49 (H9, m, 7.14, 6H). NMR C (d ¼ ppm, CDCl ): 175.0 (C7, 1C), 146.1 (C1,
o
3
1
3
H), 1.23 (H10, m, 4H), 0.92 (H11, t, J ¼ 6.42, 6H). C NMR (d 1C), 142.5 (C5, 1C), 132.6 (C2, 1C), 118.7 (C3, 1C), 114.7 (C4, 1C),
ppm, CDCl ): 177.3 (C7, 1C), 148.2 (C1, 1C), 126.5 (C2, 1C), 109.5 (C6, 1C), 47.1 (C8, 1C), 34.5 (C9, 2C), 19.9 (C10, C2), 13.4
26.0 (C5, 1C), 122.4 (C4, 1C), 120.4 (C3, 1C), 118.4 (C6, 1C), 48.0 (C11, 2C).
¼
3
1
(
C8, 1C), 35.4 (C9, 2C), 20.8 (C10, 2C), 14.1 (C11, 2C).
N-(2-Hydroxy-5-chlorophenyl)-2-propylpentanamide (VA
6
ꢀ1
).
,
ꢁ
N-(2-Hydroxy-5-methylphenyl)-2-propylpentanamide (VA
2
ꢀ1
). Dark brown solid, yield 42.4%, mp 90–92 C. FTIR (n ¼ cm
ꢁ
Dark brown solid, yield 95.7%, mp 99–101 C. FTIR (n ¼ cm
,
KBr): 3650–2400 (O–H), 3251 (N–H), 3185 (C
2
–H), 2958, 2932,
sp
KBr): 3580–2400 (O–H), 3252 (N–H), 3221, 3033 (C –H), 2957, 2876 (Csp –H), 1630, 1593 (–C(]O)–N–), 1541, 1524 (–C(O)]N–),
2
3
sp
1
2
1
8
9
927, 2872, 2861 (Csp
3
–H), 2660, 2341, 1630, 1605 (–C(]O)–N–) 1291, 867, 821 (C–Cl). NMR H (d ¼ ppm, CDCl ): 8.17, 7.86
3
541, 1507 (–C(O)]N–), 1358, 1317, 1267, 1259, 1034, 1015, 949, (OH, s, 1H & NH, s, 1H), 7.40 (H3, s, 1H), 6.95 (H5, d, 1H), 6.95
1
68, 821, 780, 766, 727, 713. NMR H (d ¼ ppm, CDCl
.20, 8.94 (OH, sb, 1H & NH, s, 1H), 7.37 (H3, s, 1H), 6.83–6.81 (H10, m, 4H), 0.92 (H11, t, J ¼ 6.60, 6H). NMR C (d ¼ ppm,
): 177.3 (C7, 1C), 147.0 (C1, 1C), 127.0 (C5, 1C), 126.3 (C2,
3
, J ¼ Hz): (H6, d, 1H), 2.35 (H8, q, J ¼ 6.60, 1H), 1.45 (H9, m, 4H), 1.20
13
(
(
H5, H6, m, 2H), 2.48 (H8, q, J ¼ 7.3, 1H), 2.24 (H12, s, 3H), 1.55 CDCl
3
H9, m, 4H), 1.36 (H10, m, 4H), 0.92 (H11, t, J ¼ 7.3, 6H). NMR 1C), 125.0 (C4, 1C), 121.9 (C3, 1C), 119.5 (C6, 1C), 48.3 (C8, 1C),
1
3
C (d ¼ ppm, CDCl ): 175.9 (C7, 1C), 145.5 (C1, 1C), 128.8 (C4, 35.5 (C9, 2C), 21.0 (C10, C2), 14.2 (C11, 2C). Suitable mono-
3
1
4
C), 125.8 (C2, 1C), 125.8 (C5, 1C), 122.0 (C3, 1C), 117.2 (C6, 1C), crystals for the X-ray diffraction analysis were obtained in a 4 : 1
7.3 (C8, 1C), 35.2 (C9, 2C), 20.5 (C10, 2C), 20.4 (C12, 1C), 15.9 hexane/ethyl acetate solvent mixture.
(
C11, 2C). Suitable monocrystals for the X-ray diffraction anal-
ysis were obtained in a 4 : 1 hexane/ethyl acetate solvent Light brown solid, yield 35.7%, mp 125–127 C. FTIR (n ¼ cm
mixture. KBr): 3560–2600 (O–H), 3297 (N–H), 3186 (Csp –H), 2958, 2935,
). 2867 (Csp –H), 1633, 1593 (–C(]O)–N–), 1531, 1495 (–C(O)]N–)
7
N-(2-Hydroxy-4-chlorophenyl)-2-propylpentanamide (VA ).
ꢁ
ꢀ1
,
2
N-(2-Hydroxy-4-methylphenyl)-2-propylpentanamide (VA
3
ꢀ1
3
ꢁ
1
Light brown solid, yield 92.7%, mp 68–70 C. FTIR (n ¼ cm
,
919, 896, 807 (C–Cl). NMR H (d ¼ ppm, CDCl , J ¼ Hz): 8.50,
3
KBr): 3700–2600 (O–H), 3238 (N–H), 3202, 3046 (C
2
–H), 2959, 7.95 (OH, s, 1H & NH, s, 1H), 7.18 (H3, d, J ¼ 8.40, 1H), 7.00
sp
o
2
1
7
1
929, 2875, 2858 (Csp
3
–H), 1639, 1611 (–C(]O)–N–), 1598, 1546, (H6, d, J ¼ 2.04, 1H), 6.80 (H4, dd, J ¼ 8.40, J ¼ 2.04, 1H),
m
o
m
510 (–C(O)]N–), 1469, 1459, 1322, 943, 897, 872, 815, 796, 771, 2.36 (H8, q, J ¼ 6.60, 1H), 1.47 (H9, m, 4H), 1.37 (H10, m, 4H),
1
13
60, 733. NMR H (d ¼ ppm, CDCl
3
, J ¼ Hz): 8.90, 8.45 (OH, sb, 0.92 (H11, t, J ¼ 6.60, 6H). NMR C (d ¼ ppm, CDCl
3
): 177.4 (C7,
H & NH, s, 1H), 7.16 (H3, d, J
o
¼ 7.02, 1H), 6.82 (H6, s, 1H), 6.65 1C), 149.1 (C1, 1C), 131.9 (C5, 1C), 124.8 (C2, 1C), 123.1 (C3, 1C),
(
H4, d, J
o
¼ 7.02, 1H), 2.49 (H8, q, J ¼ 6.72, 1H), 2.27 (H12, s, 120.6 (C4, 1C), 119.3 (C6, 1C), 48.4 (C8, 1C), 35.5 (C9, 2C), 21.0
3
6
1
1
H), 1.48 (H9, m, 4H), 1.29 (H10, m, 4H), 0.92 (H11, t, J ¼ 6.72, (C10, C2), 14.3 (C11, 2C).
1
3
H). NMR C (d ¼ ppm, CDCl
3
): 177.0 (C7, 1C), 148.4 (C1, 1C),
36.9 (C5, 1C), 123.4 (C2, 1C), 122.3 (C3, 1C), 121.1 (C4, 1C),
19.4 (C6, 1C), 47.9 (C8, 1C), 35.4 (C9, 2C), 20.8 (C12, 1C), 20.0
Semiempirical determination of partition coefficient (log P)
A partition coefficient (log P) calculation was developed in
Hyperchem v.8.0 computational chemistry soware. This was
performed for VPA and VA1–7, starting from the X-ray structures
obtained herein and through the replacement of particular
functional groups and substituents for the whole series. Mini-
mization was carried out with the PM3 semiempirical approach
for these means and log P was computed for each compound at
this level of theory.
(
C10, 2C), 14.2 (C11, 2C). Suitable monocrystals for the X-ray
diffraction analysis were obtained in a 4 : 1 hexane/ethyl
acetate solvent mixture.
N-(2-Hydroxy-5-nitrophenyl)-2-propylpentanamide
4
(VA ).
ꢁ
ꢀ1
Light yellow solid, yield 24.6%, mp 85–87 C. FTIR (n ¼ cm
,
KBr): 3500–2400 (O–H), 3416 (N–H), 3097 (Csp –H), 2957, 2937,
2
2
872 (Csp
3
–H), 2362, 2344, 1627, 1595 (–C(]O)–N–), 1539, 1503
(–C(O)]N–), 1465, 1461, 1427, 1400, 1341 (–N(]O)–O), 1327,
1
1
268, 1244, 1197, 1167, 1153, 993, 878, 819, 781, 743. NMR H (d
ppm, CDCl ¼ 2.74,
, J ¼ Hz): 9.22, 8.86 (OH, sb, 1H & H3, d, J
H), 8.82 (NH, s, 1H), 7.88 (H5, dd, J
¼ 8.96, J
H6, d, J
¼ 8.96, 1H), 2.52 (H8, q, J ¼ 7.14, 1H), 1.50 (H9, m, Modied Eagle's F12 Medium (DMEM-F12, Gibco, Thermo
Cell culture and drug treatment
¼
3
m
1
o
m
¼ 2.74, 1H), 6.98 Human U373 and rat C6 cells were cultured in Dulbecco's
(
4
¼
o
1
3
H), 1.38 (H10, m, 4H), 0.92 (H11, t, J ¼ 7.14, 6H). NMR C (d Fisher Scientic) with 10% fetal calf serum and 1% penicillin/
ppm, CDCl ): 176.3 (C7, 1C), 153.4 (C1, 1C), 140.2 (C4, 1C), streptomycin in a humidied atmosphere containing 5% CO
3
2
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ꢁ
at 37 C. Due to drug hydrophobicity, the drug stocks were drugs is paracetamol or acetaminophen (APAP), which has
dissolved in Tween 80 (1 : 80 weight ratio) and then in DMSO analgesic properties and a chemical structure that contains
(
1 : 11 v/v). C6 and U373 cells were generously donated by Drs a para-aminophenol moiety and an acetyl group attached as an
24
Jos ´e Segovia and Jos ´e Antonio Arias-Monta n˜ o, both from CIN- amide to the aromatic ring. Considering the latter, it is evident
VESTAV M ´e xico.
that the incorporation of fragments such as aminophenols in
diverse structures provides special characteristics in drugs and
prototypes, thus signifying a promising way to design new VPA
derivatives.
Cell viability
2
The cells were seeded at a density of 15 625 cells per cm in a 96
well plate. The C6 cells were treated for 24 h with the drugs at
different concentrations, while the U373 cells were treated for Molecular design of VPA analogues of valproic amide (VA)
8 h. Viability was determined using WST-1 reagent (Roche) and containing aminophenols
the absorbance was measured at 440 nm in a plate reader
Innite M1000, Tecan). The cell viability was used to obtain the
4
If one considers the use of aminophenols to generate an amidic
functionality, the nitrogen atom is employed for the peptidic
bond, leaving the OH untouched and thus recovering this group
that is present in the VPA. The incorporation of OH should be as
close as possible to the amide functional group, and the ortho-
aminophenol (oAMPOH) would provide this requisite, thus
generating the desired parent compound that connects VPA
with oAMPOH. With the inclusion of an aromatic ring in
oAMPOH, it is also possible to include structural modications
with substituents that are electro-donating or electro-
(
inhibitory concentration at 50% (IC50) of the tested compounds
in the C6 and U373 cells. At least three independent assays were
performed, and in each assay triplicates were included. The IC50
determination was done by non-linear logarithmic curve tting
using GraphPad Prism version 6.00 for Windows (GraphPad
Soware, La Jolla California USA, http://www.graphpad.com).
Statistical analysis
One-way ANOVA, followed by Dunnett's multiple comparisons withdrawing in nature, in order to modulate a plausible bio-
test were performed also using GraphPad Prism v. 6.00.
logical response and determine what substitution enhanced the
activity. These variations include fragments with a slight effect
on electronic density (–H), electro-donating methyl (–Me),
25
Results and discussion
2
electro-withdrawing nitro (–NO ) and chlorine (–Cl), which has
Synthetic proposal for valproic amides (VA)
an electro-withdrawing nature but also has the capacity for pi
retrodonation to the aromatic ring.
In this research, the molecular modications performed on the
VPA parent were aimed on the acidic side, maintaining the
valproyl moiety in an attempt to preserve the VPA biological
activity. This is because acidic side modications have been
shown to cause a variety of effects that enhance the therapeutic
capacity of VPA, e.g., the simplest VPA amide, valproyl amide
Synthesis of VA1–7
To obtain the designed compounds, VPA was reacted with SOCl
in a 1 : 1 ratio in acetonitrile to yield valproyl chloride (VPCl),
2
18
which was reacted again to achieve amide formation. Once the
VPCl was formed, the corresponding oAMPOH1–7 was added to
form a 1 : 4 mixture with triethylamine to neutralize the
hydrochloric acid obtained during both synthetic steps. In this
way, it was possible to generate the amidic bond that joined VPA
with oAMPOH1–7 to obtain seven N-(2-hydroxyphenyl)-
(
VA), which despite having antiepileptic activity has been
20
demonstrated to lack iHDAC activity. Consequently, the
exchange between acidic and amidic functionalities is not
enough to produce the desired effect or even maintain it,
because the iHDAC activity is suppressed for VA. Anyway, the
preparation of other valproic amides is forthcoming, with the
aim of preserving iHDAC activity as well as other particular
biological properties like cell viability.
A common strategy in pharmaceutical design has been the
inclusion of aminophenols to generate an amidic functionality,
where the nitrogen atom forms a peptidic bond or derived
groups, in order to form new chemical chimeras through
21
22
molecular design. For example, in 2011, Yamazaki et al.
patented a benzoxazole generated through aminophenols and
intermediary amides. Their invention refers to a pharmaceu-
tical composition containing this active ingredient to treat
illnesses such as hyperlipidemia, atherosclerosis, diabetes and
complications, inammation, and heart disease. Another
23
example, patented in 2013 by Casado-Centellas et al., is
a catechol-O-methyltransferase inhibitor (iCOMT) that again is
a benzoxazole derived from aminophenols and intermediary
amides, employed in the prevention and treatment of
Scheme 1 Synthetic procedure for obtaining VA1–7. The numbering
scheme employed was followed for the NMR and X-ray
amyloidosis. Moreover, one of the most commonly employed characterization.
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ꢀ
1
valproamides (VA1–7) (Scheme 1) in yields of 24.6–95.7%. In all 896 and 807 cm for VA
7
(see Experimental section for further
cases, the completion of the reaction was followed by FTIR details).
1
analysis, due to the change of the carbonyl band from acid to
Compounds VA
were also characterized using H (Table
1–7
1
3
13
1
valproyl chloride and nally to valproyl amide. Thin layer 1), C (Table 2) and HETCOR ( C– H) NMR experiments.
chromatography of the crude products was also performed. All Multiplicities resulted according to their substitution patterns.
1
13
compounds were characterized using FTIR, H, C and HET- The HETCOR spectra results were very useful to ascertain the
1
COR NMR, and melting point analysis. Also, VA
2
, VA
3
and VA
6
unequivocal assignment of resonances in VA1–7, both for the H
1
3
were characterized by their single crystal X-ray diffraction and C spectra. Quaternary carbons, secondary amide and
structures.
phenol signals were conrmed as not having correlations in the
HETCOR experiments. Protons H8, H9, H10 and H11, which
belong to the valproyl moiety, appear in the ranges of 2.52–2.35
(H8), 1.55–1.45 (H9), 1.38–1.20 (H10) and 0.92 ppm (H11), these
resonances are slightly inuenced by the function of distance
among aromatic and valproyl fragments as well as by the
Spectroscopic and structural analysis of VA1–7
In this section, the experimental and spectroscopic evidence for
the obtained VA type compounds through Fourier transform
infrared (FTIR) spectroscopy is presented. For the case of VA ,
a single band at 3256 cm is characteristic of the N–H bond
present in a secondary amide (R–NH–C]O). A very broad band
1
3
particular substituents at the aromatic ring. In the case of
C
1
ꢀ
1
NMR, the carbonyl signal C7 appears at 177.4–175.0 ppm,
characteristic of a secondary amide with a double hydrocarbon
chain, and is slightly inuenced by aromatic substitution. In the
case of C1, ipso to OH, the peak appears in the range of 153.4–
145.5 ppm, clearly evidencing the para electro-withdrawing
ꢀ
1
is observed around 3600–2600 cm , which is characteristic of
a phenolic O–H bond. A band belonging to Csp –H from an
aromatic ring at 3077 cm and bands due to Csp –H at 2957,
929 and 2873 cm belonging to the valproyl moiety were
2
ꢀ
1
3
ꢀ1
4
effect of the –NO2 substituent in VA , as well as the –Cl
2
substituent for VA
C2 position, which is ipso to the amide, appears at 132.6–
23.4 ppm, and is mostly affected by the para –NO in VA . The
7
, which is instead in the meta position. The
identied. The bands of the amidic group appear doubled, and
could be due to two possibilities of conformers. These bands
appear since the amide and phenolic OH interact, (a)
PhꢀOH/O ]CꢀNH and (b) PhꢀðHÞ O/HꢀNꢀC]O, gives four
1
2
5
remnant C3, C4, C5 and C6 positions appear at 123.1–116.8,
140.2–114.4, 142.5–120.8, and 119.5–109.5 ppm, respectively.
Moreover, out of the seven synthesized VA compounds,
suitable monocrystals for three of them were obtained from
ethyl acetate/hexane solvent mixtures. These cases were (crys-
ꢀ
1
amidic bands in 1626, 1603, 1545 and 1497 cm . Analogously
to VA
1
, the FTIR band assignment was done for the remaining
5
and VA ,
N-(2-hydroxyphenyl)-valproamides (VA2–7). For VA
4
ꢀ
1
there were also NO
the C–Cl bands appear at around 867 and 821 cm for VA
2
bands at 1400 and 1341–1346 cm . Finally,
ꢀ
1
talline systems and space groups) VA (orthorhombic: P2 2 2 ),
, and
3
1 1 1
6
VA
2
and VA
6
1
(both as monoclinic: P2 /c), for which crystallo-
graphic data were determined through single crystal X-ray
diffraction and analysis. Rened data and structures are pre-
sented in Tables 3 and 4. Fig. 1 shows the molecular perspec-
tives following the same numbering as that used in the
spectroscopic assignment. Selected structural parameters are
shown in Table 4. These are aimed to analyse the amidic
junction between VPA and oAMPOH, including bond distances
1
a
Table 1 H NMR of compounds VA1–7
Cmpd OH NH H3 H4 H5 H6 H8 H9 H10 H11 H12
VA
1
VA
2
VA
3
VA
4
VA
5
VA
6
VA
7
8.95 8.73 7.44 6.95 7.02 6.83 2.46 1.49 1.23 0.92
9.20 8.94 7.37 6.83 6.81 2.48 1.55 1.36 0.92 2.24
8.90 8.45 7.16 6.72 6.72 2.49 1.48 1.29 0.92 2.27
9.22 8.82 8.86
9.07 8.35 8.20 7.76
8.17 7.86 7.40
8.50 7.95 7.18 6.80
—
—
—
˚
ꢁ
ꢁ
—
7.88 6.98 2.52 1.50 1.38 0.92
7.63 2.43 1.48 1.36 0.92
6.95 6.95 2.35 1.45 1.20 0.92
7.00 2.36 1.47 1.37 0.92
—
—
—
—
(A), bond angles ( ) and dihedral angles ( ) for these three
analogous structures VA , VA and VA (for which data will
—
2
3
6
—
appear in this same order in this section). A very important
observation is that aside from the attached substituent, these
three amides are very similar in conformation. This behaviour
clearly indicates a structural consensus depending on the
—
a
The numbering employed is shown in Scheme 1. Samples were
. d ¼ ppm.
dissolved in DMSO-d
6
13
a
Table 2
C NMR of compounds VA1–7
Cmpd
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
VA
1
VA
2
VA
3
VA
4
VA
5
VA
6
VA
7
148.2
145.5
148.4
153.4
146.1
147.0
149.6
126.5
125.8
123.4
126.7
132.6
126.3
124.8
120.4
122.0
122.3
116.8
118.7
121.4
123.1
122.4
128.8
121.1
140.2
114.7
125.0
120.6
126.0
125.8
136.9
120.8
142.5
127.0
131.9
118.4
117.2
119.4
116.0
109.5
119.5
119.3
177.3
175.9
177.0
176.3
175.0
177.3
177.4
48.0
47.3
47.9
47.8
47.1
48.3
48.4
35.4
35.2
35.4
35.1
34.5
35.5
35.5
20.8
20.5
20.0
20.6
19.9
21.0
21.0
14.1
13.9
14.2
14.0
13.4
14.2
14.3
—
20.4
20.8
—
—
—
—
a
The numbering employed is shown in Scheme 1. Samples were dissolved in DMSO-d
6
. d ¼ ppm.
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a
6
Table 3 Crystallographic data for compounds VA
2
, VA
3
and VA
VA
Compound
VA
2
VA
3
6
Molecular formula
Molecular
C
15
H
23NO
2
C
15
H
23NO
2
C
14
H
20ClNO
2
249.34
249.34
269.76
ꢀ
1
weight [g mol
]
Crystalline system
Space group
Monoclinic
P2 /c
Orthorhombic Monoclinic
P2 P2 /c
1
2
1 1
2
1
1
Cell dimensions
˚
a [A]
8.9236(5)
8.7861(4)
19.7125(10)
102.535(3)
1508.69(13)
4
8.5924(17)
12.938(3)
14.250(3)
90
1584.1(6)
4
9.0054(4)
8.7908(4)
19.4618(9)
102.377(3)
1504.88(12)
4
1.191
0.249
6.31 to 55.16
222/18
10 105
˚
b [A]
˚
c [A]
ꢁ
b [ ]
3
˚
Volume [A ]
Z
ꢀ3
r (calc.) [g cm
]
1.0977
0.072
1.0455
0.069
ꢀ1
m [mm
q range [ ]
Parameters/restraints 207/30
Collected reections
Independent
reections
R (int.)
]
ꢁ
2
8.42 to 56.80 9.74 to 55.10
218/30
22 986
3584
22 042
3457
Fig. 1 X-ray diffraction structures for (a) VA , (b) VA3 and (c) VA6
2
3348
(ellipsoids at 50% of probability).
0.1127
1.032
0.0517
1.091
0.0294
1.029
GOOF
belonging to the phenol and the latter being the carbonyl
R
0.0681
0.1490
0.0558
0.1151
0.0551
0.1469
˚
R
w
fragment, with bond distances of 1.75, 1.68 and 2.01 A, where
a
these values are smaller than the sum of the van der Waals radii
Numbers in parentheses are std. deviations.
˚
˚
26
(
(
1.2 A for hydrogen and 1.52 A for oxygen). The bonding angles
ꢁ
O1–H1a/O2) for this same interaction are 160, 158 and 160 ,
ꢁ
tending towards 180 due to the strong directionality between
these two fragments. Meanwhile, the torsion angles (O1–H1a/
O2–C7) present for this hydrogen bond are 65, 41 and 58 ,
characteristics of those attached moieties, and will be discussed
as follows. For these three structures, there are clear hydrogen
bond interactions between O1–H1a/O2–C7, the former
ꢁ
evidencing a needed angularity to avoid steric hindrance
between the aminophenol and valproyl moieties. This nding of
hydrogen bonding correlates with the observation in the FTIR
spectra, where four amidic bands instead of only two were
identied, as is common in simpler amides. One pair may be
due to the non-hydrogen bonded structure and the other may be
due to the hydrogen-bonded counterpart.
a
Table 4 Structural X-ray data for VA
2
, VA
3
and VA
6
Compound VA
2
VA
3
VA
6
˚
Bonding distances (A)
O1–H1a
0.91 (3)
1.75 (3)
0.83 (2)
1.247 (2)
1.338 (3)
1.429 (3)
1.392 (3)
1.362 (3)
0.99 (4)
1.68 (4)
0.83 (3)
1.246 (3)
1.331 (3)
1.424 (3)
1.388 (4)
1.364 (3)
0.67 (3)
2.01 (3)
0.82 (2)
1.247 (2)
1.335 (2)
1.426 (2)
1.392 (2)
1.356 (2)
O2/H1a
N1–H1
For the covalent behaviour of the structures, the bond
˚
distances of O1–H1a and N1–H1 were 0.91, 0.99 and 0.67 A for
C7]O2
˚
the former, and 0.83, 0.83 and 0.82 A for the latter, showing
C7–N1
N1–C2
bond contractions only for VA . In all cases, the double bonds
6
between C7 and O2 are practically the same at 1.247, 1.246 and
C2–C1
C1–O1
˚
1.247 A, evidencing a negligible substituent effect through the
structure. This last observation is reinforced, as C7–N1 also
ꢁ
Bonding angles ( )
˚
remains almost constant at 1.338, 1.331 and 1.35 A for these
O2–C7–N1
C7–N1–C2
N1–C2–C1
C2–C1–O1
H1a–O1–C1
H1–N1–C2
O1–H1a/O2
122.0 (2)
127.56 (19)
123.4 (2)
123.4 (2)
105.7 (18)
115.7 (13)
160 (3)
121.9 (2)
127.6 (2)
123.3 (2)
122.9 (2)
105.0 (2)
112.8 (18)
158 (4)
121.76 (15)
127.13 (15)
123.45 (16)
123.60 (16)
107 (2)
compounds. As examples of the general structural behaviour,
the remaining bond distances, bond angles and torsion angles
almost behave the same for the amidic junction between VPA
and oAMPOH. In particular, the amidic fragment is practically
planar with dihedral angles for N1–C7–O2–C8 of 178.5, 179.0
116.2 (14)
160 (3)
ꢁ
and 178.4 . Meanwhile, the dihedral angles for the C1–C2–N1–
ꢁ
ꢁ
C7 fragment are 45.4, 44.3 and 46.8 , clearly tending to mini-
Dihedral angles ( )
H1a–O1–C1–C2
O1–H1a/O2–C7
H1–N1–C2–C1
H1–N1–C7–O2
N1–C7–O2–C8
C1–C2–N1–C7
ꢀ33.2 (18)
65 (7)
ꢀ148.6 (15)
ꢀ176.2 (15)
178.5 (4)
45.4 (3)
ꢀ39 (3)
ꢀ36 (3)
mize the steric hindrance among the VPA and oAMPOH
moieties.
41 (11)
58 (9)
ꢀ144.9 (19)
ꢀ148.8 (15)
ꢀ175.8 (15)
178.4 (4)
46.8 (3)
ꢀ173 (2)
ꢀ179.0 (4)
44.3 (4)
Cell viability of VA1–7
a
For the cell viability tests, all the VA1–7 compounds were pre-
Numbers in parentheses are std. deviations.
dissolved in TWEEN 80 in 1 : 9 (w/w) ratios. This mixture was
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reformulated using 1 : 11 (v/v) TWEEN 80/DMSO ratios due to the modication of the base compound VA
1
with an electro-
the hydrophobicity of these derivatives, and the nal concen- donating –Me substituent in the meta or para position relative
trations were related to the respective VA compound. According to the amide group indeed increased the cell viability of the base
to the employed ratios of TWEEN 80 and DMSO, the tested compound to 41.8 ꢂ 8.4 mM for VA (28.2 times more activity
2
concentrations were below the toxicity levels stated for these than VPA) and 55.8 ꢂ 11.0 mM (21.1 times more activity than
3 2 3
substances in cell viability assays, and blank tests were also VPA) for VA in C6 cells. In U373 cells, VA and VA also showed
2
7,28
carried out.
At the tested concentrations there were no toxic an increased effect on cell viability (151.8 ꢂ 14.4 and 161.8 ꢂ
effects. For VPA and all VA1–7 compounds, their cell viabilities in 11.2 mM). This result clearly indicates that a bigger structure
C6 (rat glioma) and U373 (human glioblastoma) lines were causes an important enhancement in the cell viability inhibi-
measured. The results show that all VA compounds presented tion. Then, the compounds with an electro-withdrawing group,
more activity than VPA (see Table 5), hence indicating that the such as the –NO substituent at the meta or para position rela-
2
molecular design of these derivatives was clever in generating tive to the amide, presented a similar effect on the cell viability
new molecules that fullled the desired goal. The cell viability of of C6 cells (67.9 ꢂ 9.7 mM for VA
VPA was measured as the control, and the inhibitory concen- VA and VA . The effect of these amides was more pronounced
tration at 50% (IC50) obtained for C6 cells (1179 ꢂ 130.8 mM) in U373 cells for the compounds with –NO (64.9 ꢂ 9.0 mM for
and for U373 cells (936.5 ꢂ 174.4 mM) was similar to that of VA and 39.7 ꢂ 4.4 mM for VA ). Finally, for both cell lines, VA
previous reports, requiring a millimolar concentration for and VA were the most active compounds. They contain –Cl as
8 h. Meanwhile, for the VA1–7 compounds, the required the substituent, behaving in a pi electro-donating/sigma electro-
4
and 45.3 ꢂ 12.3 mM for VA
5
) as
2
3
2
4
5
6
29
7
4
concentrations to achieve IC50 ranged from 19.7 to 74.8 mM for withdrawing manner, placed in the para or meta position rela-
C6, and 1.5 to 212.8 mM for U373 cells, which is comparable tive to the amide. For C6 cells, the IC50 for VA was 19.7 ꢂ 4.7
6
3
0
with other designed compounds aimed for cancer treatment.
mM and 21.8 ꢂ 2.7 mM for VA
7
; in U373 cells, the doses to obtain
the IC50 were 24.6 ꢂ 2.2 mM for VA
6
and 15.5 ꢂ 2.2 mM for VA
7
.
This is mainly due to the fact that a chlorine atom attached at an
aromatic ring prevents a wide variety of chemical reactions,
enhancing the possible interactions of the compound with
Structure activity analysis of VA1–7
1
VA is the base compound and it was found to be 15.8 times
more active than VPA in C6 cells (74.8 ꢂ 8.4 mM) and 4.4 times
32
biological receptors. Aromatic moieties possessing chlorine
and halogen substituents in general) are a key pharmacological
more active than VPA in U373 cells (212.8 ꢂ 12.3 mM), in the
(
31
same range as other VPA amidic derivatives. In this case the
molecular design gained credibility, due to the fact that this
simple derivative augmented the biological response due to the
recovery of the hydrogen-bonding group nearby, where it was
originally placed in VPA. Furthermore, this modication not
only recovers this interaction due to the presence of the
aromatic ring, the biological effect is also enhanced by at least 4
times in U373 cells and almost 16 times in C6 cells. These
results clearly show that the inclusion of an aromatic ring in
this side of the parent molecule is a key method for developing
other more potent derivatives. Therefore, the inclusion of steric
and electronic modiers of this aromatic ring, as is the case for
VA2–7, should provide insight into the types of preferred inter-
actions in a given molecular receptor in the cell. In this trend,
15
feature justied because these fragments are present in an
important amount of already tested and used pharmaceutical
drugs and pesticides, and because of the plausible interactions
that they present among them, such as R–Cl/C]O, R–Cl/H–
R, R–Cl/Ar, R–Cl/Cl–R, etc.
Moreover, in order to track important physicochemical
properties responsible for the biological ndings, a semi-
empirical (Hyperchem v.8.0) partition coefficient (log P) calcu-
lation was developed. In this line, log PVPA was 2.61, log P for
1
VA was 3.39, for VA2–3 was 3.85, for VA4–5 was 3.34 and for VA6–7
was 3.90. This calculation was carried out for each compound to
track hydrophobic/hydrophilic ratios and correlate them with
biologic trends. The results clearly evidence that the most
hydrophobic ratio, resulting from the amide in VA6–7, led to the
most potent inhibition, and this was attained due to the –Cl
substituent.
Table 5 IC50 of VA1–7 measured by cell viability in glioma cell lines C6
a
In general, VA , VA and VA presented a greater effect on C6
1
2
3
and U373
4 5 6
cells than U373 cells, but VA VA , VA and VPA required similar
Compound
Substituent*
C6 (mM)
U373 (mM)
doses for the IC50. From the analysis of this data we can conclude
that the new compounds not only maintain the effect of VPA on
the cell viability, but can also achieve the same viability with
lower doses. Related references clearly state that amides of VPA
VA
1
VA
2
VA
3
VA
4
VA
5
VA
6
VA
7
–H
m-Me
p-Me
74.8 ꢂ 8.4
41.8 ꢂ 8.4
55.8 ꢂ 11.0
67.9 ꢂ 9.7
45.3 ꢂ 12.3
19.7 ꢂ 4.7
21.8 ꢂ 2.7
1179 ꢂ 130.8
212.8 ꢂ 12.3
151.8 ꢂ 14.4
161.8 ꢂ 11.2
64.9 ꢂ 9.0
33,34
m-NO
2
do not undergo hydrolysis
or further biotransformations, due
p-NO
m-Cl
p-Cl
—
2
39.7 ꢂ 4.4
to the steric hindrance of the valproyl moiety not only under
chemical but also biological conditions. Therefore, this kind of
compound should have a major effect on cell viability due to the
newly obtained chemical structure and not due to the VPA
metabolite. This demonstrates that the new base structure (with
the neutral substituent) can be modied to increase the activity,
24.6 ꢂ 2.2
15.5 ꢂ 2.2
VPA
936.5 ꢂ 174.4
a
experiments. Glioma cells were in contact with each compound (VA1–7
in complete medium for 48 h. *Position relative to the amidic group.
Data represents median ꢂ standard error of at least three independent)
2
and the best activity was found with –Cl, followed by –NO and
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nally with –Me in U373 cells. But in C6 cells, there was not
Paper

3 R. W. Johnstone, Nat. Rev. Drug Discovery, 2002, 1, 287–299.
4 M. Dokmanovic, C. Clarke and P. A. Marks, Mol. Cancer Res.,
2007, 5, 981–989.
5 D. Marchion and P. M u¨ nster, Expert Rev. Anticancer Ther.,
2007, 7, 583–598.
a signicant difference between –NO and –Me groups. This may
2
reect different histone deacetylase expression or regulation in
these cell lines and further work is under development on this
issue.
6
M. Mottamal, S. Zheng, T. L. Huang and G. Wang, Molecules,
015, 20, 3898–3941.
2
Conclusions
7
A. V. Krauze, S. D. Myrehaug, M. G. Chang, D. J. Holdford,
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2
)
or pi electro-donating/sigma
The identity of such derivatives was evidenced through spec- 10 H. Deubzer, B. Busche, G. R ¨o nndahl, D. Eikel, M. Michaelis,
1
13
troscopic characterization using FTIR, H, C and HETCOR
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three of the derivatives (VA , VA and VA ) it was feasible to 11 G. G. Mackenzie, L. Huang, N. Alston, N. Ouyang,
2
3
6
determine their chemical crystalline structures, in all cases
behaving in a similar fashion and with very similar conforma-
K. Vrankova, G. Mattheolabakis, P. P. Constantinides and
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,
,
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VA
VA
2
and VA
3
affected C6 cells more than U373 cells, but VA
4
5
, VA
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derivatives, and the chlorine-bearing molecules produced the
71, 3–8.
best results. In the biological trend –Cl is then followed by –NO2 17 O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard
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2
since they have almost the same hydrophobic/hydrophilic ratio 19 N. Pariente-Cohen, M. Weitman, N. Tania, D. T. Major,
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perspective is that VA
6
and VA
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may become lead structures in
2003, 9, 199–216.
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2
2 Y. Yamazaki, T. Toma, M. Nishikawa, H. Ozawa, A. Okuda,
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Acknowledgements
We acknowledge CONACyT projects 47310337 (L. Arregui) and
2
22872 (H. I. Beltr ´a n), UAM and SEP-PROMEP for nancial
support. A. Alpuche-Garc ´ı a and X. D ´a vila-Gonz ´a lez acknowledge 23 M. Casado Centellas, B. R. Insa, B. N. Reig and B. N. Gavald `a ,
university scholarships from CONACyT and UAM.
New therapy for transthyretin-associated amyloidosis, Spain
Pat., WO2013060668 A1, 2013.
2
4 J. A. Clements, R. C. Heading, W. S. Nimmo and
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